key: cord- - vnthmfn authors: guo, xichao; chen, yu; li, xuefen; kong, haishen; yang, shigui; ye, bo; cui, dawei; wu, wei; li, lanjuan title: dynamic variations in the peripheral blood lymphocyte subgroups of patients with pandemic h n swine-origin influenza a virus infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vnthmfn background: novel influenza a (h n ) is an acute respiratory infectious disease. animal experiments indicated that when h n virus infected early hosts, it showed strong cd (+), cd (+), and cd (+)cd (+ )t cell reactions. the aim of this study was to investigate the dynamic fluctuations of the peripheral blood lymphocyte subgroups in patients infected with h n swine-origin influenza a virus (s-oiv). methods: the frequency of t cells, b cells, natural killer (nk) cells, and regulatory t cells (treg) in severe h n and moderate h n patients were detected at different periods by flow cytometry. in parallel, serum cytokines were detected by enzyme-linked immunosorbent assay and c-reactive protein (crp) was analyzed through an image-type automatic biochemical analyzer. in addition, healthy volunteers, who were not infected with h n virus, were selected as controls. results: the frequency of nk cells were decreased in all cases and cd (+ )b cells were increased in severe cases than those of the controls. at - d from onset, the frequency of cd (+ )and cd (+)cd (+ )t cells in moderate cases was higher than in the severe cases. serum cytokines, specifically il- , il- , il- , il- , and ifn-γ exhibited no significant change both in the moderate and the severe cases during the whole monitoring process. in the early stage of the disease, serum crp levels in the severe and moderate groups were significantly higher than that in the control group. conclusions: patients showed different lymphocyte subgroup distributions between mild and severe cases, which might affect the incidence and development of h n . the latest public data from the chinese ministry of health showed , confirmed h n cases and deaths were reported from the chinese mainland as of january , . data analysis suggests that patients with chronic diseases, obese patients, and pregnant women are more liable to develop severe h n influenza. after initial infection by the virus, the host innate immune system, as a first line of defense, takes protective measures earlier than the adaptive immune system to avoid further viral invasion or replication [ ] . animal experiments indicated that when the swine-origin influenza a virus (s-oiv) infected early hosts, it elicited strong cd + , cd + , and cd + cd + t cell reactions [ ] . cd + and cd + t cells are related to viral immunity and the lack of these cells would lead to a delay in viral clearance and an increase in mortality [ ] [ ] [ ] . on the other hand, cd + cd + cells are t cell subsets with an immune inhibition function and could play an important regulatory role in a variety of infectious diseases [ ] [ ] [ ] . these complex molecular and cellular mechanisms are helpful in controlling and eliminating the acute stage of viral infection. although successfully eliminating the virus is * correspondence: ljli@zju.edu.cn † contributed equally state key laboratory for diagnosis and treatment of infectious diseases, first affiliated hospital, school of medicine, zhejiang university, hangzhou, , pr china full list of author information is available at the end of the article essential, the response of virus-specific t cells to the hosts could also result in tissue damage and autoimmune responses. therefore, monitoring the variation of cellular immune functions in h n patients have important clinical significance. the aim of this study is to analyze the dynamic fluctuations of t cells, b cells, natural killer (nk) cells, and regulatory t cells in patients infected with novel influenza h n , as well as serum cytokines and c-reactive protein (crp). up to h n outpatients and inpatients were chosen from august to december in the department of infectious diseases, first affiliated hospital, school of medicine, zhejiang university. among these, cases were severe and cases were moderate (table ). in addition, healthy volunteers who were not infected with h n virus, were selected as controls. there were no statistical differences between their ages and genders compared with the h n patients. the diagnostic criteria for h n were based on the influenza a h n flu clinic program (pilot version of the second edition, ) released by the chinese ministry of health in july . its main content includes the following: i. confirmation standard of influenza a (h n ) cases: patients are considered to have contracted the virus if they present with influenza-like clinical manifestations and had one or more of the following laboratory test results: ( ) positive for s-oiv nucleic acid (through real-time rt-pcr and rt-pcr); ( ) s-oiv is isolated and attained; and ( ) specific neutralizing antibody titers for serum s-oiv are increased four times or more. ii. severe cases: when confirmed or suspected cases manifest one of the following signs, they are considered as severe: ( ) complicated pneumonia with or without hypoxemia and respiratory failure; ( ) toxic shock; and ( ) multiple organ dysfunction syndrome or multiple organ failure. the study protocol was approved by the ethics review committee of the first affiliated hospital, school of medicine, zhejiang university. all subjects understood the procedures and consented participate in the study. on day , , , , , , , , and , ml of whole blood (edta-k anticoagulant) and . ml of serum were collected from h n patients for the detection of lymphocyte subgroups, cytokines, and crp. the same sample collection was carried out in the control group. afterwards, μl of anticoagulant was added into all flow tubes and μl of cd -pc , cd -fitc, cd -pe, cd -fitc-cd( + )-pe, cd -fitc, and cd -pe mouse anti-human fluorescence monoclonal antibodies (bd bioscience) was added. after mixing, the samples were incubated for min away from light at room temperature. red blood cell lysis and cell fixation using coulter qprep specimen processing instrument (beckman coulter inc., usa) was conducted, and the cells more than × were counted through a flow cytometer (coulter cytomics fc , beckman coulter). the results were expressed as the percentages of cd + cd + , cd + , and cd + t cells, as well as those of cd + b cells and nk (marked as cd -cd + cd + ) cells found positive for the antigen marker in the total lymphocytes population. the data were collected and analyzed using the expo software (beckman coulter inc., usa). serum crp detection was conducted using the imagetype automatic biochemical analyzer (beckman coulter inc., usa). the kit was also provided by the same company. serum cytokines, namely il- , il- , il- , il- , and ifn-γ were tested using an enzyme-linked immunosorbent assay (elisa; ex-cell). the experimental methods were in accordance with the instructions of the manufacturer in the reagent kit. statistical analysis spss . statistical package was used for data processing. measurement data were indicated as mean ± standard deviation or percentages. comparisons among groups were carried out using one-way anova or χ test. differences with p < . were considered statistically significant. correlation analysis among the detection indicators were conducted using spearman's rank correlation analysis. up to patients with confirmed laboratory diagnoses of influenza were enrolled in this study. the baseline characteristics of the patients are summarized in table . the samples were predominantly composed of full-grown adults with s-ovi infection. as shown in table , the patients with severe h n were significantly older than those that presented with moderate h n (mean age, . years vs. . years; p < . ). the mean duration of symptoms before presentation was days, with fever, cough, and myalgia as the most common in both groups. however, the patients with severe h n had more prodromal symptoms (e.g., fever, dyspnea, and myalgia) and co-morbidities [e.g., chronic obstructive pulmonary disease (copd) and coronary artery disease] than the patients with moderate h n (p < . ). although a relationship with pregnancy was more common in the severe h n group (four patients vs. one patient with moderate h n ), this difference was not statistically significant (table ) . similarly, there was no difference in the other preexisting diseases such as diabetes mellitus and hypertension between the two groups. chest x-rays were taken from all patients, and abnormalities were detected in patients, with moderate h n and with severe h n . ten patients presented with radiological abnormalities attributable to chronic lung conditions without evidence of concurrent pneumonia. up to patients with severe h n were admitted to the intensive care unit (icu) with preexisting diseases, such as copd. all patients were treated with oseltamivir (tamiflu) and % of the severe h n patients were additionally treated with glucocorticoids ( / ). the duration of oseltamivir treatment was based on the pandemic influenza a (h n ) clinical guidelines (second edition, ). however, the subjects were not given immunomodulators. treatment response did not differ significantly between the two groups and the mean duration of hospital confinement was . days (range, - days) and . days (range, - days) for the severe and moderate patients, respectively. overall, only one severe h n patient died during the course of the study. no patient dropped out and all completed the -week follow-up. the frequency of peripheral blood cd + b cell count of patients with severe h n gradually increased within the first - d of treatment. the nk cell count showed a gradual decline, whereas the t cell subtypes (cd + cd + , cd + , and cd + t cells) showed no significant changes. the frequency of peripheral cd + t and cd + cd + treg cells of patients with moderate h n were significantly higher than in patients with severe h n within the first - d of treatment. the nk cell counts of patients with moderate h n were similar to that of patients with severe h n , which demonstrated a gradual decline, whereas the frequency of cd + b and cd + t cells showed no significant changes. the frequency of the different lymphocyte subsets at different periods were statistically compared and the results show that the frequency of peripheral cd + and cd + cd + t cells in the moderate h n patients were higher than that of the severe h n patients within the first - d (figures a and c ; p < . ). the cd + b cell counts of the severe h n patients were significantly higher than those of the moderate h n patients and the control group for the same period (figure e) , whereas the nk cell counts were significantly less than that of the control group (figure d ; p < . ). the frequency of cd + t cells had no significant difference (p > . ; figure b) among the three groups. in the first - d of study, the serum crp levels of both the patients with severe h n and the patients with moderate h n were significantly higher than that of the control group (p < . ; figure f ). correlation analysis of the cd + , cd + t, cd + cd + , cd + b, and nk cells with crp was conducted and the results indicate that the various lymphocyte subgroups had no significant correlations with crp. the serum cytokines of patients, specifically il- , il- , il- , il- , and ifn-γ, whether the h n infection was severe or moderate, showed no significant changes during the whole monitoring process (data not shown). the differences of the h n patients with the control group had no statistical significance. influenza a (h n ) is the greatest pandemic threat in [ ] . the clinical course of h n can be severe, particularly for very young patients with complications. the immune response affected by pathological damage to the body caused by s-ovi and clinical prognosis. studies have shown that the incidence of patients with severe h n was highest in the -to -year-old age group and the lowest for patients aged or above [ ] . our clinical data also showed that the most severe infections occur in individuals younger than years old or in middle-aged patients figure analysis of the dynamic changes in the lymphocyte subgroup and c-reactive protein (crp) in the peripheral blood of patients with influenza a (h n ) . cd + t, cd + t, cd + cd + treg, nk, and cd + b cells are presented as a percentage of total peripheral blood leukocytes (pbls) in severe h n (n = ) patients, moderate h n (n = ) patients, and healthy volunteers (n = ). †: denotes statistically significant differences compared with severe h n ; *: denotes statistically significant differences compared with the healthy volunteers; **: denotes statistically significant differences compared with the moderate h n patients and with the healthy volunteers. older than years old with comorbidities, especially lung disease. based on local guidelines, antiviral treatment would be considered for severe or critical cases and high-risk patients infected with the influenza a pandemic (h n ) virus within h of onset. for patients who presented later than days, the managing physicians were allowed to make decisions regarding antiviral use. oseltamivir, given to patients older than years, was prescribed according to the standard dosing regimen ( mg twice daily orally, for days). dosage adjustment, whenever necessary, were based on the patient's renal function ( mg daily, if creatinine clearance is < ml/min). for children ( - years old), dosage adjustments were based on body weight (bw), that is: mg twice daily for children with bw < kg; mg twice daily for bw - kg; mg twice daily for bw - kg; and mg twice daily for bw > kg. accordingly, patients with acute respiratory distress syndrome (ards) were treated with high dose glucocorticoids over a short period of time, which has been proven beneficial and safe. several lines of experimental evidence suggest that h n infections accompanied by a characteristic impairment of the innate immune response. by monitoring the host functional response, patients with increased risks of developing severe influenza-associated complications could be identified immediately [ , ] . the preliminary data from this study showed that the frequency of cd + b cells in patients with severe h n was significantly higher than in the moderate and control groups (figure e) , and the frequency of nk cells in the severe and moderate groups were less than that of the control group. in the host innate immune system, nk cells are the major effector cells during acute infection, rapidly killing infected cells in the process. previous studies have shown that the significant decrease in peripheral nk cell count is mainly because the s-ovi directly infected and killed nk cells, thereby limiting their activity and leading to their apoptosis in vivo [ ] . animal studies have found that cd + t, cd + t, and cd + b cells achieved peak values within - d after s-ovi infection, whereas the cd + cd + treg cell count reached its peak at - h after infection [ ] . currently, the use of flow cytometric analysis to measure peripheral blood cd + , cd + , and cd + cd + t cells is a simple and common method. reportedly, the mostly cd + cd + t cells were also positive for foxp [ ] . in this experiment, the peak values for all lymphocyte subgroups in patients with severe h n could not be detected. at least two mechanisms could account for the lower effector t cell response detected after the s-ovi infection, and the peak response time for the effector t cells possibly missed. in addition, the virus could have also led to the immunotolerance of the t cells [ ] . as intercellular signaling molecules, cytokines regulate the immune response, mediate inflammatory responses, and participate in tissue repair. experiments have shown that only the peak values of ifn-α and il- could respectively be detected within and h in the serum of infected animals, as well as in the plasma of pediatric patients with severe influenza [ , ] . in this experiment, no statistically significant changes were found during the continuous monitoring of serum il- , il- , il- , il- , and ifn-γ of patients with severe and moderate h n , as well as in the control group. this result might also be related to the immunotolerance of t cells, of which the peak detection might have been missed. the mechanisms responsible for maintaining these relatively constant levels remain unclear. crp is an acute phase protein produced by the liver and is a sensitive marker for the inflammatory response. it did not directly related to viral load [ ] . however, crp continuously increased in the initial stages of infection for both the patients with severe and those with moderate h n . the crp of patients with severe h n took longer to return to normal, which might be the result of underlying disease. each lymphocyte subgroup has made correlatively analyzed with crp and the results show no significant correlations. in conclusion, s-oiv could stimulate the cellular immune response. when accompanied by cd + b cell increase and a continuous nk cell decline, it could indicate that the body is in a dynamic balance of the antiinfection immunity and autoimmune damage; this is particularly true for patients with severe influenza. each lymphocyte subgroup in patients with h n plays a more important antiviral role in the early stages of disease, but excessive immune response also leads to the increase and development of infection. innate immune response to viral infection pathogenesis and transmission of the novel swine-origin influenza virus a/h n after experimental infection of pigs rapid recovery of lung histology correlates with clearance of influenza virus by specific cd + cytotoxic t cells transgenic mice lacking class i major histocompatibility complex-restricted t cells have delayed viral clearance and increased mortality after influenza virus challenge clearance of influenza virus respiratory infection in mice lacking class i majorhistocompatibility complex-restricted cd + t cells natural regulatory t cells in infectious disease regulatory t cells: friend or foe in immunity to infection? treg control of antimicrobial t cell responses world health organization: influenza a (h n ): who announces pandemic alert phase , of moderate severity surveillance of the first confirmed hospitalised cases of pandemic h n influenza in ireland association of profoundly impaired immune competence in h n v-infected patients with a severe or fatal clinical course effect of the novel influenza a (h n ) virus in the human immune system influenza virus directly infects human natural killer cells and induces cell apoptosis cells with regulatory function of the innate and adaptive immune system in primary sjögren's syndrome t-cell tolerance for variability in an hla class i-presented influenza a virus epitope immune dysregulation in severe influenza cytokines and acute phase proteins associated with acute swine influenza infection in pigs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution authors' contributions xg and yc performed the majority of experiments and drafted the manuscript. xl and hk followed-up the patients and involved in editing the manuscript. sy and ww did most of clinical works. by and dc provided analytical tools. ll was the principal investigator and provides all facilitates to complete this work. all authors read and approved the final manuscript. key: cord- -vzbh k authors: zhang, weijun; lin, yan; bai, yu; tong, tiegang; wang, qun; liu, nihong; liu, guangliang; xiao, yihong; yang, tao; bu, zhigao; tong, guangzhi; wu, donglai title: identification of cd (+ )cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vzbh k twenty-seven nanopeptides derived from the matrix (m) protein of porcine reproductive and respiratory syndrome virus (prrsv) were screened for their ability to elicit a recall interferon-γ (ifn-γ) response from the splenocytes of balb/c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus rwr-prrsv-m. we identified two peptides (amino acid residues k( )fitsrcrl and f( )gymtfvhf) as cd (+ )cytotoxic t lymphocyte (ctl) epitopes. these peptides elicited significant numbers of ifn-γ secreting cells, compared with other m nonapeptides and one irrelevant nonapeptide. bioinformatics analysis showed that the former is an h- k(d)-restricted ctl epitope, and the latter is an h- d(d)-restricted ctl epitope. multiple amino acid sequence alignment among different prrsv m sequences submitted to genbank indicated that these two ctl epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to prrsv. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine viral pathogens, and has caused significant economic losses to the swine industry worldwide. characterization of field isolates suggested that prrsv are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. based on significant sequence differencesprrs viruses are grouped into two distinct genotypes, european isolate (lelystad virus, lv) and north american isolate (vr- ) [ ] prrsv has two major structural proteins, gp and m, encoded by orfs and , respectively. gp , the most important neutralizing antigen of prrsv, has the highest genetic diversity among isolates [ ] . and, recent studies in yorkshire × landrace crossed and outbred pigs, showed that there are two immuno-dominant t-cell epitopes derived from the gp protein: l aalicfvirlaknc and k grlyrwrspvii/vek [ ] . the m protein, which contains highly conserved amino acid sequences, also has very good immunogenicity and is associated with protection against prrsv infection. dna vaccinations have also revealed that m is the most potent inducer of t lymphocyte proliferation [ ] . at present, effective vaccination strategies for the prevention and control of prrsv infection are not available. vaccines based on inactivated prrsv virus have been ineffective at inducing protective immune responses. live-attenuated prrsv vaccines can provide protection against this pathogen, but have been observed to revert to virulence [ ] , restricting the application of this vaccination approach. the rational development of future prrsv vaccines will necessitate a systematic understanding of the protective humoral and cellular immune responses that occur during prrsv infection, and should aim to induce a broad immune responses that accommodates the plasticity of the major antigenic sites. recent research has indicated that cell-mediated immunity may play a very important role in the clearance of prrsv [ ] . major histocompatibility complex (mhc) i restricted cytotoxic t lymphocytes (ctls) can kill virus-infected cells and eliminate potential sources of new virus [ ] . hence, identification of ctl epitopes is crucial in the design of synthetic vaccines, and a number of studies have successfully identified pathogen-derived t cell epitopes [ ] [ ] [ ] [ ] . unlike many other pathogens, there is limited knowledge of the specific prrsv-derived peptides targeted by t-cells. in the current study, we report the identification of ctl epitopes from the prrsv (ch- a strain) m protein in a mouse model. we screened peptides derived from the prrsv m protein for their ability to induce interferon (ifn)-γ in splenocytes harvested from balb/ c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus expressing m protein. the screen identified two peptides that elicited ifn-γ production in cd + cd + splenocytes of vaccinated mice. a multiple amino acid sequence alignment among different prrsv m proteins indicates that these two peptides are strongly conserved across multiple prrsv strains and therefore should be considered for further research. the prrsv ch- a strain, the vaccinia virus wr strain, and the akabane virus obe- strain were part of our laboratory collection. the former was propagated in marc- cells, and the latter two were propagated in bhk- cells, respectively, and these two cell lines were cultured in dmem (invitrogen) supplemented with % of fetal calf serum (fcs, invitrogen) in a humidified °c, % co incubator. total rna was extracted from the prrsv ch- a strain and the spleens of balb/c mice. full length cdnas were amplified based on the complete open reading frames (orfs) of m and ubiquitin (ub) following reverse transcription (genbank accession numbers ay and az ) using the m-f and m-r primers pair or the ub-f and ub-r primer pair ( table ) . the full length cdnas were cloned into the pmd -t vector (takara) and are referred to as pmd-m and pmd-u, respectively. an indirect enzyme-linked immunosorbent assay (ielisa) method was established to evaluate specific antibody against m protein. a truncated m protein ( - aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. the truncated m gene cdna was amplified from pmd-m using the truncated-m-f and truncated-m-r primers (table ) , ligated into pgex- p- (ge healthcare), and subsequently named pgex-m. for protein expression, pgex-m was transformed into e.coli bl (de ) and recombinant protein expression was induced with . mm iptg. the samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline (pbs, ph . ). after being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and western blot with anti-prrsv-m monoclonal antibody (our laboratory collection), the fusion-expressed truncated m protein was purified by gst tag according to the manufacturer's instructions (ge healthcare). finally, the concentration of the purified protein was determined using a bradford kit (bio-rad) according to the manufacturer's instructions. the ub and m genes were fused using splicing by overlapping extension pcr (soe-pcr). the ub and m genes were amplified from pmd-u and pmd-m with the primer pairs ub-f and fusion-m-ub-r and fusion-m-ub-f and m-r, respectively ( table ). the fusion gene product, referred to as ub-m, was amplified from the purified pcr products with ub-f and m-r and inserted into the eukaryocyte expression vector pvax , and was named pvax -ub-m. the transferring vector psc (our laboratory collection), which is composed of the early promoter p . and late promoter p of vaccinia virus, and the lacz and amp genes controlled by the promoter p as the reporter genes, was used in this study. to construct the transferring vector psc -m, the complete m gene was amplified with the psc -m-f and psc -m-r primer pair (table ) and inserted into the psc . the primer p . -f (table ) , derived from promoter p . sequence, was used for directional identification and the positive clones, which were subsequently named psc -m. homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid psc -m and the wr strain vaccinia virus into % confluent tk - cells cultured in mem medium containing μg/ml brdu (sigma). the viruses were collected after the appearance of cytopathic effect (cpe), and recombinant vaccinia virus was purified according to the expression of lacz gene. western blot analysis was performed as described previously [ ] . briefly, a bhk- cell layer was infected with rwr-prrsv-m recombinant vaccinia virus or the vaccinia virus wr strain at a multiplicity of infection (moi) of . . cells were harvested days post-infection, and total cell lysates were prepared with lysis buffer ( mm tris-cl ph . , mm mgcl , . % np , μg/ml dnase i). cell lysates were separated by sds-page and were subsequently transferred to a membrane for western blot analysis using an anti-prrsv-m monoclonal antibody, hrp-conjugated goat anti-mouse igg secondary antibody (bio-rad), and dab substrate. the positive recombinant virus was named rwr-prrsv-m. the expression of m was further confirmed by ifa. briefly, bhk- cells were infected with either rwr-prrsv-m or the vaccinia virus wr strain when the bhk- cells reached - % confluence in a -well plate. m protein expression was evaluated by ifa days post infection using the anti-prrsv-m monoclonal antibody followed by a fitc-conjugated rabbit antimouse igg secondary antibody (sigma). specific fluorescence was observed using a fluorescence microscope (leica dm ire ). the sequences of m were screened for potential h -k d / h -d d /h -l d -mer epitopes using the algorithms from the syfpeithi website [ ] , the hla peptide binding prediction website (bimas) [ ] and pred balb/c [ ] . the peptides (table ) , with highest binding score (bs) as predicted by each algorithm, were synthesized by scilight biotechnology llc (beijing, china) and purified to a purity > % using high performance liquid chromatography (hplc). all animal experiments were performed according to national and institutional guidelines. one hundred and ninety female balb/c mice (vital river laboratory animal technology co., beijing, china) were maintained in isolation cages at the experimental animal center of harbin veterinary research institute (harbin, china). mice were divided into three groups: the pvax -ub-m vaccination group (n = ), the pvax control vaccination group (n = ) and the pbs control vaccinated group (n = ). the plasmid dna used for immunization was purified using the endofree mega plasmid preparation kit (qiagen). the pvax -ub-m and pvax cohorts were intramuscularly (i.m.) vaccinated with μg pvax -ub-m or pvax plasmid dna in μl pbs, respectively. the pbs control group received an i.m. injection of μl pbs. each group was vaccinated four times at -week intervals. to enhance the specific ctl responses to m protein, the mice received an intraperitoneal (i.p.) injection containing . moi of rwr-prrsv-m on day following the fourth dna vaccination. at the same time, five mice from the pvax and pbs vaccination groups were also inoculated intraperitoneally with . moi of rwr-prrsv-m on day following the fourth vaccination in order to compare the specific antibody raised against m protein of different experimental groups following vaccination with rwr-prrsv-m. all procedures were conducted with the protocols approved by experimental animal center of harbin veterinary research institute (hvri) of the chinese academy of agricultural sciences (caas). to investigate the specific antibody response, serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m. an ielisa was performed according to methods described previously [ ] . the purified m protein was used as the coating antigen, the tested sera applied at a : dilution, and an hrp-conjugated goat anti-mouse igg antibody (bio-rad) used as the secondary antibody. the microplates were developed using orthophenylene diamine (opda, amersco) and h o for min, after which the reaction was stopped by the addition of m h so . finally, the optical density (od) was read at nm. an anti-prrsv-m monoclonal antibody was used as a positive control. serum containing antibodies against akabane virus and the sera of control group mice served as negative controls. isolated splenocytes were added to u-bottomed -well plates (corning inc) at cells/well in μl complete rpmi- medium supplemented with % fcs. the cells were then mixed with μl media containing note: the peptides with highest binding score (bs) as predicted by each algorithm are shown, starting with the peptide giving the highest score at the top for each protein. sequences of mers are given from bimas, syfpeithi and pred balb/c predictions, respectively. the n-terminal amino acid position is indicated for epitopes predicted from the whole m protein. a nonamer peptide derived from prssv m protein at μg/ml, or phorbol- -myristate- -acetate (pma ng/ ml) and ionomycin ( ng/ml) as a positive control. cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (ibv) h strain [ ] were used as negative controls. following a h incubation at °c, um monensin (sigma) was added and the splenocytes were incubated for an additional h at °c before staining. the number of cd + cd + t cells producing ifn-γ on days , and after boosting with rwr-prrsv-m was determined using flow cytometric analysis. ics analysis was performed using a facscalibur flow cytometer (bd) according to the methods described previously [ ] . twenty mice were tested from the group vaccinated with pvax -ub-m, and five mice from the groups vaccinated with pvax and pbs were tested at each time point. the splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with m protein-derived peptides, as detailed in section . . ten thousand cd + t lymphocytes were acquired per sample and the number of ifn-γ-secretion cd + cd + t cells were enumerated. the cd + cd + lymphocytes that expressed ifn-γ following peptide stimulation were considered to be peptide-specific ctls. specific ctl responses were evaluated as the increase in the number of cd + cd + ifn-γ + cells. reagents used include percp-conjugated cd e, pe-conjugated cd a and fitc-conjugated ifn-γ, and bd cytofix/cytoperm™, all purchased from becton, dickinson & co (bd). to further confirm the results of the ics, the other sixty mice from the group vaccinated with pvax -ub-m and thirty mice from the groups vaccinated with pvax and pbs were tested by the ifn-γ elispot assay at three different time points (as detailed in section . ). data are expressed as the number of ifn-γ-secreting cells per two hundred thousand splenocytes. peptide-specific ifn-γ elispot responses were considered to be positive if the response (minus media background) was ≥ fold above the media response and ≥ spot-forming cells (sfc)/ × splenocytes were registered. the ielisa results, the percentage of ifn-γ positive cd + cd + t lymphocytes and the number of spots per × splenocytes were analyzed using the analysis of variance (anova), and a probability value below . was considered significant. as the full-length m protein is difficult to express in vitro, we expressed a truncated version of m that lacks hydrophobic amino acids at the n-terminus. sds-page analysis showed that cells transformed with the pgex-m expression vector produced a large amount of protein with a molecular mass of approximately kda, consistent with the expected molecular weight of the truncated m protein fused with a gst tag (data not shown). western blot analysis using an anti-prrsv-m antibody confirmed the expression and identity of the truncated m protein fused with a gst tag (additional file , fig. s ). the ub proteasome pathway (upp) is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. in order to enhance the ub-mediated degradation efficiency of m protein, we expressed a ub-m fusion protein in bhk- cells using the eukaryotic expression plasmid pvax -ub-m, in which the ub coding sequence was fused in-frame with the prrsv m coding sequence. following transient transfection of bhk- cells with the pvax -ub-m plasmid, the ub-m fusion protein was expressed and accumulated predominantly in the cytoplasm (additional file , fig. s ). immunization strategies that prime and boost with recombinant dna vectors encoding antigens have been shown to elicit t-cell immunity against hiv in non-human primates [ ] , and more recently, in humans [ ] . therefore, we used a dna vector encoding the prrsv m protein to immunize mice in order to generate and characterize m protein-reactive cd + t cells. the recombinant vaccinia virus rwr-prrsv-m drove expression of an m protein with the expected molecular weight when transfected into bhk- cells (additional file , fig. s ). ifa confirmed expression of m protein following transfection of bhk- cells, and revealed m protein accumulation in the cytoplasm (additional file , fig. s ). m protein-specific serum antibody increased steadily from the second to the fourth dna vaccination with the pvax -ub-m vector which drives m protein expression in vivo (additional file , fig. s ), indicating that dna vaccination elicited m protein-specific immune responses as expected. a subsequent booster vaccination with the recombinant vaccinia virus, rwr-prrsv-m, elicited a further increase in prrsv-specific antibody (p < . , additional file , fig. s ). in contrast, control mice vaccinated with pvax -only or pbs-only did not show significant increases in m protein-specific antibody titers after the booster vaccination with rwr-prrsv-m (p > . , additional file , fig. s ). overall, mice vaccinated with pvax -ub-m generated significantly higher m protein-specific antibody titers compared to mice vaccinated with pvax or pbs (p < . , additional file , fig. s ). these results indicate that rwr-prrsv-m amplifies the protective effects of dna vaccination and reveals the advantage of this priming-boosting strategy. dna vaccination with pvax -ub-m likely drives the differentiation of memory b cells which are subsequently activated by rwr-prrsv-m following the booster immunization, resulting in increased m-specific antibody titers. mice vaccinated with pbs and pvax would not be expected to generate m protein-reactive memory b cells, accounting for a less pronounced increase in m protein-specific antibody titers following rwr-prrsv-m innoculation. in this study we identified potential ctl epitopes in balb/c mice vaccinated with pvax -ub-m and boosted with rwr-prrsv-m. the frequency and number of cd + cd + t cells that produced ifn-γ following stimulation with peptides derived from prssv m protein was enumerated using ics and elispot assays. using these approaches, we identified two peptides from prssv m protein that elicited ifn-γ production from the splenocytes of vaccinated mice. as shown in figure , intracellular ifn-γ staining following stimulation of splenocytes from vaccinated mice with the peptide k fitsrcrl and f gymtfvhf revealed a population of ifn-γ producing cd + t cells comprising - % of the total cd + splenocyte population. in contrast, unstimulated splenocytes and splenocytes exposed to an irrevelant peptide did not contain a population of ifn-γ producing cd + t cells (figure , panel b and c). consistent with the ics data, peptides k fitsrcrl and f gymtfvhf elicited ifn-γ production from splenocytes of vaccinated mice when measured by elispot, whereas unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide did not reveal ifn-γ producing cells (figure ). the k fitsrcrl and f gymtfvhf prssv m protein peptides were identified bioinformatically as h- k d and h- d d restricted ctl epitopes (table ) . specific increases in the number of cells producing ifn-γ following stimulation with the peptides "k fitsrcrl" and "f gymtfvhf" was observed by day after the booster vaccination with rwr-prrsv-m (figure and ) . furthermore, splenocytes from mice vaccinated with pvax -ub-m responded strongly to "k fitsrcrl" and "f gymtfvhf", but not other m protein-derived peptides, at all time points tested following the booster vaccination with rwr-prrsv-m. when the same pattern of reactivity on three different time points after boosting with rwr-prrsv-m within each vaccination group were analyzed statistically using anova analysis, statistically significant differences were noted for the peptides "k fitsrcrl" and "f gymtfvhf" when compared to the other peptides tested among mice vaccinated and boosted with pvax -ub-m and rwr-prrsv-m, respectively (p < . , figure a and b). conversely, and confirming the specificity of these responses, no difference among the stimuli was observed with mock-vaccinated (pvax ) mice and naïve mice (data not shown). it is important to recognize that the ics assay calculates the percentage of ifn-γ + cells among cd + t cells in the spleen ( - %), whereas the elispot assay assesses the number of ifn-γ + cells among all splenocyte cell types ( . %), and cannot definitively assign the production of ifn-γ to a particular cell type. thus, the two assays use different denominators in calculating the frequency of ifn-γ + production by splenocytes. importantly, each method clearly identified k fitsrcrl and f gymtfvhf as the only two peptides from a panel of that elited significant ifn-γ production from the splenocytes of vaccinated mice. in this study we used balb/c mice as a model system to identify ctl epitopes in the m protein of prssv to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. the identification of prssv ctl epitopes in balb/c mice allow for the future generation of reagents, such as mhc class i: peptide staining reagents, that will enable the in-depth investigations of cd + t cell responses during prssv infection and immuinization. whether these two epitopes can bind the sla class i molecules of pigs remains to be determined. in some cases, specific peptide epitopes are known to be recognized by cytotoxic t cells in different animal species. for instance, the core region of hcv contains an epitope that is recognized by cytotoxic t cells of both mice and humans [ ] . further research on the role of these peptide epitopes in different species is ongoing in our laboratory. in conclusion, we identified peptides "k fitsrcrl" and "f gymtfvhf" from the m protein of prssv as h- k d and h- d d restricted ctl epitopes, respectively. in this study, we also developed a mouse model of prrsv infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to prrsv. porcine reproductive and respiratory syndrome virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates identification of immunodominant t-cell epitopes present in glycoprotein of the north american genotype of porcine reproductive and respiratory syndrome virus t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations immunological responses of swine to porcine reproductive and respiratory syndrome virus infection hepatitis b virus immunopathogenesis understanding presentation of viral antigens to cd + t cells in vivo: the key to rational vaccine design identification of an h- d(b)-restricted cd + cytotoxic t lymphocyte epitope in the matrix protein of respiratory syncytial virus characterization of a new h- d(k)-restricted epitope prominent in primary influenza a virus infection dna immunization with c fmdv non-structural protein reveals the presence of an immunodominant cd +, ctl epitope for balb/c mice immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp / gp of porcine reproductive and respiratory syndrome virus and swine il- syfpeithi: database for mhc ligands and peptide motifs scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains predbalb/c: a system for the prediction of peptide binding to h d molecules, a haplotype of the balb/c mouse dna vaccination of pigs with open reading frame - of prrs virus localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus a dna/mva-based candidate human immunodeficiency virus vaccine for kenya induces multi-specific t cell responses in rhesus macaques enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution additional file : elisa antibody response in mice after immunization following dna vaccination and a booster vaccination with recombinant vaccinia virus. fig.s . m protein-specific antibody responses in mice immunized with pbs, or pvax or pvax -u-m dna, and boosted with rwr-prrsv-m. serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m, and were evaluated for reactivity to mprotein in an elisa based on coating with the truncated m protein fused with a gst tag. and, the day represents the day of the first dna immunization. statistically significant differences are indicated by "*" or "**" for p-values < . or < . , respectively, as determined by anova.authors' contributions dlw and gll gave me the idea of this study. wjz and yl participated in the design and conducted the majority of the experiments as well as drafted the manuscript. tgt helped revise the manuscript and participated in the first stage of the experiments. yb and qw participated in the prediction of ctl epitopes and analyzed the data. yhx, nhl and ty participated in the sequence alignment. zgb provided the expression system of recombinant vaccinia virus, and gzt provided the prrsv ch- a strain. all the authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -t y b gb authors: sumibcay, laarni; kadjo, blaise; gu, se hun; kang, hae ji; lim, burton k; cook, joseph a; song, jin-won; yanagihara, richard title: divergent lineage of a novel hantavirus in the banana pipistrelle (neoromicia nanus) in côte d'ivoire date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: t y b gb recently identified hantaviruses harbored by shrews and moles (order soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. to investigate this possibility, archival tissues from insectivorous bats (order chiroptera) were analyzed for hantavirus rna by rt-pcr. following numerous failed attempts, hantavirus rna was detected in ethanol-fixed liver tissue from two banana pipistrelles (neoromicia nanus), captured near mouyassué village in côte d'ivoire, west africa, in june . phylogenetic analysis of partial l-segment sequences using maximum-likelihood and bayesian methods revealed that the newfound hantavirus, designated mouyassué virus (mouv), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for nova virus in the european common mole (talpa europaea). full genome sequencing of mouv and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses. discovery of phylogenetically divergent hantaviruses in shrews and moles (order soricomorpha, family soricidae and talpidae) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] raises the possibility that rodents (order rodentia, family muridae and cricetidae) may not be the principal or primordial reservoirs. moreover, newfound hantaviruses harbored by soricomorphs of multiple species, distributed in widely separated geographic regions across four continents, suggest that their host diversity may be far more expansive than previously assumed. specifically, other mammals having shared ancestry or ecosystems with soricomorphs may serve as reservoirs and may be important in the evolutionary history and diversification of hantaviruses. in particular, bats (order chiroptera) may be potential reservoirs by virtue of their rich diversity and vast geographical range, as well as their demonstrated ability to host myriad medically important, disease-causing viruses [ ] [ ] [ ] [ ] [ ] . surprisingly little attention, however, has been paid to this possibility. as in our previous investigations on the spatial and temporal distribution of hantaviruses in soricomorphs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , we relied on the availability of archival tissues. using the purelink micro-to-midi total rna purification kit (invitrogen, san diego, ca), total rna was extracted from frozen and ethanol-fixed liver and other visceral tissues of insectivorous bats (representing genera), collected during may to june in asia, africa and the americas (table ) . cdna was then prepared with the superscript iii first-strand synthesis system (invitrogen) using random hexamers, and pcr was performed as described previously, using an extensive panel of oligonucleotide primers, designed on conserved genomic sequences of rodentand soricomorph-borne hantaviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . each reaction mixture contained μ dntp, mm mgcl , u amplitaq polymerase (roche, basel, switzerland) and . μ oligonucleotide primers. initial denaturation at °c for min was followed by two cycles each of denaturation at °c for s, two-degree step-down annealing from °c to °c for s, and elongation at °c for min or min s, then cycles of denaturation at °c for s, annealing at °c for s, and elongation at °c for min, in a geneamp pcr thermal cycler (perkin-elmer, waltham, ma). amplicons were purified using the qiaquick gel extraction kit (qiagen, hilden, germany), and dna sequencing was performed using an abi prism xl genetic analyzer (applied biosystems, foster city, ca). after innumerable failed attempts, hantavirus rna was detected by rt-pcr in ethanol-fixed liver tissues from two of banana pipistrelles (neoromicia nanus peters ), captured during june near mouyassué village ( ° ' "n, ° ' "w) in aboisso district, km from abidjan, in the extreme southeastern region of côte d'ivoire in west africa ( figure ). the taxonomic identity of the hantavirus-infected vesper bats was confirmed by phylogenetic analysis of the cytochrome b gene of mtdna (genbank jq ), amplified by pcr as previously described [ , ] . despite similarly exhaustive efforts, hantavirus rna was not detected in any of the other bat species tested (table ) , including frozen liver tissue of six tiny pipistrelles (pipistrellus nanulus), collected in parc national du mont péko, km northwest of mouyassué, in february , and ethanol-fixed liver tissue of three tiny pipistrelles, collected in december in azagny, where a hantavirus was previously found in the west african pygmy shrew (crocidura obscurior) [ ] . a -nucleotide region of the rna-dependent rna polymerase-encoding l segment, amplified using a hemi-nested primer set (outer: '-gaaaggg-cattnmgatgggcntca gg- ', '-aaccadt-cwgtyccrtcatc- '; inner: '-gnaaaytnatgt-atgtnagt gc- ', '-aaccadtcwgtyccrt-catc- '), was aligned and compared with hantavirus sequences available in genbank, using clustalw (dnastar, inc., madison, wi) [ ] and transalign [ ] . the newfound hantavirus, designated mouyassué virus (mouv), exhibited low nucleotide and amino acid sequence similarity of less than % to all representative soricomorph-and rodent-associated hantaviruses, except for the . % sequence similarity with nova virus (nvav), previously reported in the european common mole (talpa europaea) [ ] . interestingly, mouv sequences were identical in the two banana pipistrelles (kb and kb ), a male-female pair captured simultaneously and presumed to be a mating couple, suggesting horizontal virus transmission or common-source infection. mouv formed a uniquely divergent lineage, distant from all other hantaviruses identified to date, except for nvav (figure ), in phylogenetic trees based on l-segment sequences, generated by the maximum-likelihood and bayesian methods, implemented in paup* (phylogenetic analysis using parsimony, . b ) [ ] , raxml blackbox webserver [ ] and mrbayes . [ ] , under the best-fit gtr+i+Γ model of evolution established using jmodeltest . . [ ] . topologies were well supported by bootstrap analysis of iterations, and posterior node probabilities based on two runs each of million generations sampled every generations with burn-in of %. despite the overall success of our brute-force rt-pcr approach at identifying previously unrecognized hantaviruses in frozen tissues [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and tissues preserved in rnalater ® rna stabilization reagent [ , ] , designing universal primers for the amplification of soricomorph-borne hantaviruses has presented continuing challenges. thus, while it is likely that many more hantaviruses await discovery, overcoming technical barriers is essential to facilitating their detection. viewed in this context, the failure to detect hantavirus rna in all but one bat species was not altogether unexpected and may be attributed simply to suboptimal primer design and imperfect cycling conditions. also, low rna yields and poor rna preservation in tissues fixed in ethanol under field conditions may have thwarted our efforts at obtaining more of the mouv genome. that said, the successful amplification of hantavirus rna from ethanol-fixed tissues is highly instructive and augments the pool of archival tissues for future exploratory studies of hantaviruses in bats, and possibly other insectivorous small mammals that share ancestral lineages with soricomorphs, such as hedgehogs (order erinaceomorpha, family erinaceidae). dating to the seminal discovery of hantaan virus in lung tissue of the striped field mouse (apodemus agrarius) [ ] , lung has been the preferred tissue in studies aimed at finding new hantaviruses [ ] [ ] [ ] . however, lung is not the only tissue in which hantaviruses can be detected [ , ] . in our search of genetically distinct hantaviruses in long-stored archival tissues from shrews and moles, lung tissue was frequently unavailable. instead, liver tissue was more often accessible and proved to be quite suitable [ , , , ] . similarly, liver tissues were more often available in the present study. as in reservoir rodents and soricomorphs, hantavirus rna is likely to be present in many tissues of persistently infected bats. real-time quantitative rt-pcr analysis of lung, liver and other viscera will clarify the tissue distribution of mouv in newly captured banana pipistrelles from mouyassué. having their fossil origins in the eocene epoch, approximately million years before present, bats occur on every continent except antarctica and are among the most speciose orders of mammals, with more than , extant species [ ] . the banana pipistrelle, which is distributed widely in forests and savannas across sub-saharan africa ( figure c, inset) , is one of species in the genus neoromicia of the family vespertilionidae and subfamily vespertilioninae. like other vesper bats, the banana pipistrelle is insectivorous. unlike large fruit bats, such as the straw-colored fruit bat (eidolon helvum) and hammer-headed bat (hypsignathus monstrosus), which are sold as bush meat, the banana pipistrelle, weighing approximately g, is not consumed as food. however, because banana pipistrelles occasionally roost within houses or reside near human habitation, rare human encounters raise the possibility of hantavirus exposure. previously, serological evidence of hantavirus infection was reported in the common serotine (eptesicus serotinus) and greater horseshoe bat (rhinolophus ferrumequinum) captured in korea [ ] , but genetic analysis of hantaviral isolates from these insectivorous bat species proved to be indistinguishable from prototype hantaan virus [ ] , suggesting laboratory contamination. in the present study, the strikingly divergent lineage of mouv precluded any possibility of contamination and lends support to our earlier conjecture that the ancient origins of hantaviruses may have involved insect-borne viruses [ , ] , with subsequent adaptation to and host switching between early soricomorph and chiropteran ancestral hosts in the mammalian superorder laurasiatheria. however, since the biological and evolutionary implications of bats as reservoirs of hantaviruses are considerable, studies are underway to establish that the banana pipistrelle is the natural host of mouv. moreover, high-throughput sequencing technology is being applied to obtain the full genome of mouv and to ascertain the geographic range and genetic diversity of hantaviruses harbored by bats. novel hantavirus sequences in shrew seewis virus, a genetically distinct hantavirus in the eurasian common shrew (sorex araneus) hantavirus in northern short-tailed shrew, united states newfound hantavirus in chinese mole shrew phylogenetically distinct hantaviruses in the masked shrew (sorex cinereus) and dusky shrew (sorex monticolus) in the united states characterization of imjin virus, a newly isolated hantavirus from the ussuri white-toothed shrew (crocidura lasiura) genetic diversity and phylogeography of seewis virus in the eurasian common shrew in finland and hungary molecular evolution of azagny virus, a newfound hantavirus in the west african pygmy shrew (crocidura obscurior) in côte d'ivoire genetic diversity of thottopalayam virus, a hantavirus harbored by the asian house shrew (suncus murinus) in nepal molecular phylogeny of a newfound hantavirus in the japanese shrew mole (urotrichus talpoides) host switch during evolution of a genetically distinct hantavirus in the american shrew mole (neurotrichus gibbsii) evolutionary insights from a genetically divergent hantavirus harbored by the european common mole (talpa europaea) shared ancestry between a mole-borne hantavirus and hantaviruses harbored by cricetid rodents bats: important reservoir hosts of emerging viruses serological evidence of infection with nipah virus in bats (order chiroptera) in peninsular malaysia bats are natural reservoirs of sars-like coronaviruses swanepoel r: fruit bats as reservoirs of ebola virus recent common ancestry of ebola zaire virus found in a bat reservoir hantavirus in african wood mouse grouping of hantaviruses by small (s) genome segment polymerase chain reaction and amplification of viral rna from wild-caught rats clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice or: transalign: using amino acids to facilitate the multiple alignment of protein-coding dna sequences phylogenetic analysis using parsimony (*and other methods) a rapid bootstrap algorithm for the raxml web servers mrbayes : bayesian phylogenetic inference under mixed models posada d: jmodeltest: phylogenetic model averaging isolation of the etiologic agent of korean hemorrhagic fever nephropathia epidemica: detection of antigen in bank voles and serologic diagnosis of human infection isolation of hantaan virus, the etiologic agent of korean hemorrhagic fever, from wild urban rats genetic identification of a hantavirus associated with an outbreak of acute respiratory illness observations on natural and laboratory infection of rodents with the etiologic agent of korean hemorrhagic fever a new natural reservoir of hantavirus: isolation of hantaviruses from lung tissues of bats genomic characterization of m and s rna segments of hantaviruses isolated from bats divergent lineage of a novel hantavirus in the banana pipistrelle (neoromicia nanus) in côte d'ivoire we thank melissa s. nagata, nelson i.b. lazaga, moti l. chapagain and pakieli h. kaufusi for technical assistance. we also thank shaobin hou and the staff of the advanced studies in genomics, proteomics and bioinformatics for dna sequencing. this work was supported in part by grants r ai from the national institute of allergy and infectious diseases and p gm from the national institute of general medical sciences, national institutes of health. authors' contributions ls and shg independently performed rna extraction, rt-pcr and dna sequencing reactions. hjk and jws provided primer design and phylogenetic analysis. bk, bkl and jac provided tissues and information about bats. ry conceived the project and provided overall scientific oversight. all authors contributed to the preparation of the final manuscript. the authors declare that they have no competing interests. key: cord- -ci db d authors: guo, ling; yang, shao-lin; chen, shi-jie; zhang, zhihe; wang, chengdong; hou, rong; ren, yupeng; wen, xintian; cao, sanjie; guo, wanzhu; hao, zhongxiang; quan, zifang; zhang, manli; yan, qi-gui title: identification of canine parvovirus with the q r point mutation in the vp gene from a giant panda (ailuropoda melanoleuca) date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ci db d background: in this study, we sequenced and phylogenetic analyses of the vp genes from twelve canine parvovirus (cpv) strains obtained from eleven domestic dogs and a giant panda (ailuropoda melanoleuca) in china. a novel canine parvovirus (cpv) was detected from the giant panda in china. results: nucleotide and phylogenetic analysis of the capsid protein vp gene classified the cpv as a new cpv- a type. substitution of gln for arg at the conserved residue in cpv presents an unusual variation in the new cpv- a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus. conclusions: these findings extend the knowledge on cpv molecular epidemiology of particular relevance to wild carnivores. canine parvovirus type (cpv- ) is an important pathogen in domestic dogs and several wild carnivore species. it was first identified in usa in and was found later to have spread worldwide in domestic and wild canine populations. cpv- is a small non-enveloped, singe-stranded dna virus ( . kb) which is a member of the genus parvovirus of the family parvoviridae. the cpv genome is composed of two major orfs, one encoding the two nonstructural proteins [ns and ns ] and the other encoding the two capsid proteins [vp and vp ] [ ] . there is also a third protein, vp which is produced by proteolytic processing of vp . cpv- is antigenically and genetically related to feline panleukopenia virus (fplv). however, fpv infects cats, mink and raccoons, but not dogs, whereas cpv- infects dogs and other canidae, but not cats. a few amino acid differences between cpv and fpv determine the specificity of these viruses [ ] . after the cpv- initial appearance (during - ) , two new antigenic variants, named cpv- a and cpv- b, were characterized [ ] [ ] [ ] . the antigenic types cpv- a and cpv- b differ from the original cpv- in at least five or six amino acids/residues of the vp capsid protein (genomic positions , , , and ) [ , ] . canine parvovirus type a/ b having mutation at residue (ser→ala) is designated as new cpv- a/ b [ , ] , residue is located in a minor antigenic site close to epitope b and substitutions at this position may be responsible for changes in antigenicity of cpv variants [ ] . another antigenic variant having an amino acid substitution -asp→glu was reported for the first time in italy [ ] and had been reported from other countries [ , [ ] [ ] [ ] [ ] [ ] [ ] , and this variant is currently named as cpv- c. it has been reported that canine parvovirus (cpv) have been implicated in disease and mortality in giant pandas [ ] [ ] [ ] [ ] , which is an endangered species native to the china. the giant pandas with cpv infection showed diarrhea, vomiting and water-like feces [ ] . giant panda parvovirus vp gene described here identifies yet another variant of the virus. it demonstrates the continued adaptation of the virus to an everexpanding host range that includes endangered species of wildlife. understanding emergent disease theats is important in enabling effective conservation measures for endangered species. out of faecal samples of giant pandas and canine rectal swabs screened by pcr assay using hfor/hrev primers, giant panda and dog samples yielded a specific amplicon of bp, respectively. the amplified pcr products of randomly selected canine samples and one giant panda sample were subjected for sequencing using primer pair hfor/hrev. primer pair hfor/hrev [ ] encompasses informative amino acid residues which are of significance in characterizing the cpv types. all the cpv samples under study were found to be new cpv- a (cpv- a with nucleotide variation t→g at position or cpv- a with amino acid variation -ser→ala). in comparison to prototype new-cpv- a (ay ), the samples under this study had amino acid residue variations at tyr ile caused by mutation tat →att at nt - of the vp gene. it was a unique mutation within the vp of chinese and korean strains of "new cpv- a". critical positions of the cpv vp gene products of samples sequenced in this study are summarized in table . in addition to the nucleotide variations at positions and , three additional mutations were observed in the canine parvovirus sequences under study. one was at nucleotide position where a mutation (u→a) resulting in the codon change from uuc→uac, with amino acid variation -phe→tyr. all the sequences under this study except b , b and b showed this variation. the second one was at nucleotide position , where variation a→g was observed and which changed the codon from acg→gcg, with amino acid variation -thr→ala. this variation (a→g) at nucleotide position was observed in strains a , a , a , b , b , b , b and b in this study (the dog samples). the last mutation was at nucleotide position where a mutation (a→g) resulting in the codon change from caa→cga, with amino acid variation -gln→arg. this variation only was revealed in strain b (the giant panda sample). to analyse the phylogenetic relationships of the china isolates with other cpv strains isolated in various parts of the world, we constructed a maximum likelihood phylogenetic tree. the panda field isolate b was found to be phylogenetically closely related to new cpv- a strains of jilin strain cnjl . b and b are the closest of chinese dog sequences examined in this study. rest of the other sequences had distinct lineage but shared molecular relationship with new cpv- a reference strains ( figure ). new cpv- a/ b appear to have replaced the prototype cpv- a/ b strains and become the predominant types in many countries [ ] [ ] [ ] [ ] [ ] . in china, the prevalent antigenic type in canine populations is type a, our results seem to confirm these data [ ] . the dog and giant panda samples were all new cpv- a, but it is impossible to conclude that new cpv- a is predominant strain in the china dog or giant panda population because our data are limited to an exiguous number of samples and we do not have data regarding cpv- dog strains from the area where the giant panda samples were collected. all the cpv clinical samples under study were found to be new cpv- a ( figure ). similar substitution -ser→ala in cpv- a strain were reported in vp gene of cpv from worldwide [ , , [ ] [ ] [ ] . this study detected one site mutation among the cpv- a isolates in china: tyr→ile at position , this mutation was also reported in [ ] . previous studies have shown that residue is subject to strong positive selection in all parvoviruses of carnivores [ ] . the natural adaptation of a virus to a new host is a very rare event, suggesting that there are high barriers that prevent viruses from gaining the ability to infect and spread naturally in hosts to which they are not adapted. in this study, the novel point mutation virus is most likely not a panda-adapted virus spreading among pandas, but more likely a spill-over from dogs. because host adaptation involved complex interactions among both surface-exposed and buried capsid mutations that together altered cell infection and immune escape properties of the viruses [ ] . in this study, the virus vp gene coding change at nt (vp residue , gln→arg) was interesting because it has not been detected previously in any other strains. we speculated that the mutation has two aspect functions. on the one hand, residue was close to residues and , it has indicated that residues and are associated with the ability of cpv to hemagglutinate or alter ph dependence of hemagglutination [ ] , on the other hand, residue was adjacent to residue and , while the and residue affects canine transferrin receptor (tfr) binding to determined the canine host range [ ] . so, we made the inferences that r might be involved in a required conformational change, or it might mediate an effect on receptor binding through the neighboring residues, and is likely to have had an effect on the parvovirus host range. this observation suggests that the glutamine to arginine mutation may also affect host dna±protein interaction. as is the case for wild animal, this mutation is not selected for the population, but may have arisen independently from various backgrounds. as the phylogenetic tree shows, most of the viruses isolated in china formed a large cluster, while some strains clustered together with viruses from regions outside china. most of the cpvs isolated in china formed a specific cluster and certain mutations detected in chinese cpvs probably arose during the process of local adaptation, as indicated by previous surveys [ ] . considering that the cpv- vaccine appears to provide a comparatively lower and shorter immunity against heterologous cpvs, there is evidences to suggest that complete immunity may not be provided to dogs even if cpv- vaccines are used [ ] [ ] [ ] . in many countries, such as europe, cpv- a has been overtaken by cpv- b or cpv- c, some researches have been to evaluate antigenic relationships among the original canine parvovirus type (cpv- ) and the variants cpv- a, - b, and - c, cross-antigenic evaluation revealed clear differences among the cpv variants [ ] . nevertheless, our study showed that the giant panda and dogs are all new cpv- a type, while the vaccines available for giant pandas and dogs are cpv- type vaccine in china (nobivac®, holland) [ ] . the effectiveness of cpv- vaccine against cpv- a type has not been evaluated in china. in sum, wildlife in captive facilities in china is generally not reliably or safely vaccinated. strain difference between field virus and vaccine candidate virus could be one of the important attributable reasons for some immunization failure. infectious diseases pose a significant risk to these animals, of which many are endangered species, the mechanisms of the virus into the giant panda population and the adaptation (mutation) of the virus to that species are important topics for future research. the animal from which specimens were collected, was handled in accordance with animal protection law of the people's republic of china (a draft of an animal protection law in china released on september , ). this study was approved by the national institute of animal health animal care and use committee at sichuan agricultural university (approval number - ). a total of faecal samples of giant pandas and rectal swabs were collected from dogs suspected to be infected with cpv were gathered simultaneously. detailed information on the origin and the accession numbers of the cpv-positive samples is shown in table . all activities followed the legal requirements and institutional guidelines set out by the government of p.r. china. the samples were collected in china during a period of months from november to may . the collected samples were emulsified in ml of . m pbs of ph . and centrifuged at g for min at °c. the supernatant was collected and used for pcr amplification. hundred microlitres of the processed supernatant was used for template dna preparation by boiling at °c for min and chilling immediately in crushed ice [ , ] . the supernatants were diluted : in distilled water to reduce residual inhibitors of dna polymerase activity [ ] . primer pair and pcr amplification pcr amplification was performed using kod-plus-ver. (toyobo, japan) and primer pair h for ( ′-caggtg atgaatttgctaca- ′)/h rev ( ′-catttggata -aactggtggt- ′) that amplifies bp fragment of the gene encoding capsid protein [ ] . pcr amplification was consisted of cycles of denaturation ( °c s), annealing( °c s), extension ( °c s) and final extension ( °c min) and the products were analyzed by electrophoresis using . % agarose gel in tris acetate edta (tae) buffer ( ×). pcr products of the correct size ( bp in length) were amplified and cloned using target clone-plus-(toyobo, japan),then custom sequenced with primer pair hfor/ hrev. the sequences were aligned with sequences of prototype cpv strains (m -fpv; m -cpv- ; m -cpv- a; m -cpv- b; ay -new cpv- a; ay -new cpv- b; fj -cpv- c) using clustal w (http://www.clustal.org). the sequences were analyzed with respect to the prototype cpv- strain for the nucleotide variation of vp gene at positions , , , , and with the corresponding amino acid residues at , , , , and , respectively. for the phylogenetic analysis, canine parvovirus sequences from various parts of the world were retrieved from the genbank and used. the sequences were aligned using clustal w. maximum likelihood tree was drawn using the mega . software [ ] . canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type c prevalence and genetic characterization of canine parvoviruses in korea new approaches for the molecular characterization of canine parvovirus type strains rapid antigenic-type replacement and dna sequence evolution of canine parvovirus natural variation of canine parvovirus canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells evolution of canine parvovirus: loss and gain of the feline host immunogenicity of an intranasally administered modified live canine parvovirus type b vaccine in pups with maternally derived antibodies chronological analysis of canine parvovirus type isolates in japan evolution of canine parvovirus-a need for new vaccines? evidence for evolution of canine parvovirus type in italy first detection of canine parvovirus type c in pups with haemorrhagic enteritis in spain a novel antigenic variant of canine parvovirus from a vietnamese dog molecular characterization of canine parvovirus- variants circulating in tunisia molecular epidemiology of canine parvovirus detection of a canine parvovirus type c with a non-coding mutation and its implications for molecular characterisation occurrence of canine parvovirus type c in the united states serosurvey of infection of parvovirus in giant pandas serologic survey of giant pandas (ailuropoda melanoleuca), and domestic dogs and cats in the wolong reserve serosurvey of ex situ giant pandas (ailuropoda melanoleuca) and red pandas (ailurus fulgens) in china with implications for species conservation serosurvey of selected viruses in captive giant pandas (ailuropoda melanoleuca) in china analysis of vp gene sequences of canine parvovirus isolates in india molecular epidemiology and phylogeny reveal complex spatial dynamics in areas where canine parvovirus is endemic surveillance activity for canine parvovirus in italy recombination between vaccine and field strains of canine parvovirus is revealed by isolation of virus in canine and feline cell cultures western european epidemiological survey for parvovirus and coronavirus infections in dogs phylogenetic analysis of the vp gene of canine parvoviruses circulating in china predominance of canine parvovirus (cpv) in unvaccinated cat populations and emergence of new antigenic types of cpvs in cats analysis of canine parvovirus sequences from wolves and dogs isolated in italy genetic analysis of vp gene of canine parvovirus isolates in korea genetic analysis of canine parvovirus type c phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses the role of evolutionary intermediates in the host adaptation of canine parvovirus the three-dimensional structure of canine parvovirus and its functional implications evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs selective regimen shift and demographic growth increase associated with the emergence of highfitness variants of canine parvovirus antibody levels and protection to canine parvovirus type severe parvovirus in a -year-old dog that had been repeatedly vaccinated evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type c. microbiol-quart evaluation of the antigenic relationships among canine parvovirus type variants current status of canine distemper vaccine immune among giant pandas and red pandas clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant a simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors declare that they have no competing interests. key: cord- -qlutgfu authors: barberis, abdelheq; boudaoud, amine; gorrill, angelina; loupias, josianne; ghram, abdeljelil; lachheb, jihene; alloui, nadir; ducatez, mariette f. title: full-length genome sequences of the first h n avian influenza viruses isolated in the northeast of algeria date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: qlutgfu background: h n avian influenza viruses (aiv) has a worldwide geographic distribution and affects poultry of different types of production. h n aiv was first reported in the northeast of algeria in april , following an outbreak associated with high mortality, in broiler flocks. in the present study, we report full-length genome sequences of aiv h n , and the detailed phylogeny and molecular genetic analyses. methods: ten aiv h n strains, collected in broiler flocks, were amplified in -day-old embryonated specific pathogen free (spf) chicken eggs. their full-length genomes were successfully sequenced and phylogenetic and molecular characterizations were conducted. results: phylogenetic analysis showed that the isolates were monophyletic, grouped within the g- lineage and were very close to moroccan and algerian strains identified in and , respectively. the low pathogenicity of the strains was confirmed by the sequence motif ( rssr/glf ) at the hemagglutinin (ha) cleavage site. an exclusive substitution (t a) that had not been previously reported for h n viruses; but, conserved in some pandemic h n viruses, was observed. when compared to the g -like h n prototype, the studied strains showed one less glycosylation site in ha, but – additional ones in the stalk of the neuraminidase (na). the ha protein harbored the substitution l, suggesting binding preference to human-like receptors. the na protein harbored s a and r w substitutions, previously detected in h n from asia and the middle east, and especially in h n and h n strains that caused human pandemics. different molecular markers associated with virulence and mammalian infections have been detected in the viral internal proteins. the matrix m protein possessed the s n substitution associated with drug resistance. the non-structural (ns ) protein showed the “gsev” pdz ligand (pl) c-terminal motif and no – deletion. conclusion: characterized algerian aiv isolates showed mutations that suggest increased zoonotic potential. additional studies in animal models are required to investigate the pathogenicity of these h n aiv strains. monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. implementation of adequate biosecurity measures that limit the introduction and the propagation of aiv h n in algerian poultry farm is crucial. conclusion: characterized algerian aiv isolates showed mutations that suggest increased zoonotic potential. additional studies in animal models are required to investigate the pathogenicity of these h n aiv strains. monitoring their evolution in both migratory and domestic birds is crucial to prevent transmission to humans. implementation of adequate biosecurity measures that limit the introduction and the propagation of aiv h n in algerian poultry farm is crucial. keywords: avian influenza h n , algeria, full-length genome sequencing, phylogenetic analysis, molecular characterization background avian influenza (ai) is an infectious viral disease that mainly affects the respiratory or digestive system. the avian influenza viruses (aiv) affect different species of wild and domestic birds [ ] . they belong to the orthomyxoviridae family, having a single stranded rna genome composed of eight segments that code for more than viral proteins. the hemagglutinin (ha) and the neuraminidase (na) genes code for the major virus surface glycoproteins [ ] . based on their antigenic properties, aivs are classified into different subtypes. sixteen subtypes of ha (h -h ) and nine of na (n -n ) have been identified in birds [ ] , in different combinations. two ha (h and h ) and two na (n and n ) were identified exclusively in bats [ ] . the aiv h n was first detected in the usa, in [ ] , but now, it is considered as a worldwide distributed pathogen [ ] . the h n aiv subtype harbors two phylogenetic lineages: the north american lineage and the eurasian lineage [ , ] . the last one contains different prototypes that are grouped in at last three sub-lineages. the g -like sub-lineage is represented by the prototype virus a/quail/hong kong/g / ; the y -like sub-lineage by a/duck/hong kong/y / , a/chicken/beijing/ / , and a/chicken/hong kong /g / ; and finally, the korean-like sub-lineage by a/chicken/korea/ -p / and a/duck/hong kong/y / [ ] . despite their low pathogenicity, aiv h n viruses have led to heavy economic losses, particularly during coinfections with other respiratory pathogens [ , ] . also, it has been reported that low pathogenic avian influenza virus (lpaiv) h n can easily undergo genetic reassortment and donate internal gene segments to highly pathogenic avian influenza viruses (hpaiv) h and h [ , [ ] [ ] [ ] . in addition, different studies, showed that circulating h n strains have acquired affinity to mammalian like-receptors and gained high virulence and pathogenicity through substitutions in their viral proteins [ , ] ; the most known substitutions are in the ha protein that promotes virus binding to cellular receptors. likewise, the polymerase complex protein, which is composed of three subunits (polymerase basic "pb " protein, polymerase basic "pb " and polymerase acidic "pa" proteins), harbors multiple molecular markers, which also affect host tropism and pathogenicity of influenza viruses. similarly, the matrix (m) and the non-structural (ns) proteins also contain amino acids (aa) that contribute to the virus growth capabilities in mammalian cell cultures [ ] . currently, very limited data is available on the circulation of aivs in algeria. concerning hpaivs, the algerian veterinary services have reported, in november , the first case of h n infection in the natural park of ghardaia, in the south of algeria. the collected samples from dead migratory birds tested positive by specific quantitative rt-pcr. however, no control measures were applied [ ] , with the concern of spread of virus from migratory birds to domestic poultry. recently, jeevan et al., ( ) have reported isolation of h n aiv from an outbreak observed, in , in the central region of algeria. their study focused on phylogenetic characterization and pathogenicity of the isolates in experimental animals [ ] . however, the full-length genome sequence analysis had not been carried out. in the present study, we report the isolation and the full-length genome sequencing of aiv-h n , isolated from broiler poultry farms, in the north east of algeria (province of batna), during . genome sequences were successfully obtained for the all isolate genomes, followed by phylogenetic and detailed amino acid (aa) sequence analyses of determinants of virulence, host specificity and pathogenicity. in the third week of april , an epizootic outbreak of a fatal and highly contagious respiratory disease was reported in broiler flocks in the province of batna (north east of algeria). the mortality rate in the affected farms was variable, ranging from to %, with an average daily mortality of chickens per flock. outbreaks particularly occurred in flocks older than days of age (age of slaughter in algeria: > days). this epizootic outbreak subsequently spread very quickly to all broiler farms in the province of batna. neighboring provinces (sétif and m'sila) were also affected. at necropsy, the most observed lesions were mainly localized in the respiratory and digestive tracts, such as hemorrhages in the trachea and intestines. yellow fibrinous exudates were also found in the lumen of the trachea, with fibrinous casts in bronchi. given the constraints encountered in the field, broiler flocks affected when the outbreak was declared were sampled according to their accessibility. twenty organ pools (trachea, kidneys, liver, and intestines) were collected from april to september (two organ pools per flock); each organ pool containing samples (trachea, kidneys, liver or intestines) collected from five randomly selected broiler chickens. samples were immediately placed in cryotubes containing viral transport medium (pbs-glycerol, sigma®, antibiotics and antifungi), and then transported to the laboratory at °c, and stored at − °c until used. each organ pool was homogenized in dmem (dulbecco's modified eagle medium) (gibco, thermo fisher scientific) supplemented with % penicillin-streptomycin, and then clarified by centrifugation ( , rpm for min at + °c). the upper aqueous phase was collected for rna extraction. total rna was extracted by the trizol method (invitrogen, carlsbad, ca, usa), according to the manufacturer's instruction. avian influenza a virus was detected by real time rt-pcr, using agpath one-step kit (ambion, applied biosystems, grand island, new york) with primers and probes specific to the conserved region of the matrix (m) gene, previously described by spackman et al., ( ) [ ] . reactions were performed according to the following protocol: one cycle of °c for min and °c for min, followed by cycles of °c for s and °c for min. positive samples were then subtyped by rt-pcr, using aiv h detection kit (intron biotechnologie®, korea), using the following protocol: °c for min and °c for min, followed by cycles of °c for s, °c for s, °c for s, and a final elongation step of °c for min. the upper aqueous phases from organ pools that were aiv positive, were used to isolate the virus in -day-old specific pathogen free (spf) embryonated chicken eggs (pfie, inrae, nouzilly, france), inoculated via the allantoic route. embryonic mortality within h after inoculation was considered nonspecific. the allantoic fluid was then harvested h post-inoculation and total viral rna was extracted using the qiaamp® viral rna mini kit (qiagen®, germany), following the manufacturer's instructions. the full-length genome amplification of the studied strains was carried out by conventional rt-pcr using segment specific primers, as previously described by hoffmann et al., ( ) [ ] . the rt-pcr reaction was performed in a peqlab peqstar thermocycler (vwr), with the following program: °c for min, °c for min, followed by cycles of °c for s, °c for s and °c for . min. a final extension step at °c for min was then performed. the pcr products of the expected length were purified from the agarose gel using the qiaquick® gel extraction kit (qiagen®, germany), following the manufacturer's instructions. the purified amplicons were subjected to nucleotide sequencing using the same primers as in rt-pcr by the gatc biotech platform (germany). consensus dna sequences were constructed from forward and reverse sequences in a dna baser v . . [ ] . nucleotide (nt) and aa sequence alignments were carried out using clustalw in bioedit sequence alignment editor v . . . the blast software programs (https://blast. ncbi.nlm.nih.gov/blast.cgi) were used to determine the sequence similarity of the algerians strains. phylogenetic trees were constructed by maximum likelihood method in a mega x . . program [ ] , using the general time reversible (gtr) nucleotide substitution model; one thousand bootstrapping replicates being used to estimate the robustness of the tree branches. the nucleotides sequences of all studied strains are available in genbank database, under accession numbers summarized in table . phylogenic characterization of the studied algerians strains was performed for all their aiv gene segments, in comparison with selected aiv h n strains from genbank. for amino acid analyses, selected sequences were subjected to clustalw multiple sequence alignment [ ] . the analysis was performed for different molecular markers of host viral tropism, pathogenicity and virulence, as previously described for lpaiv h n and other subtypes. all organ pools tested to detect aiv genome were positive to aiv h n subtype. virus from of the pools could successfully be isolated in embryonnated eggs and characterized. phylogenetic analysis showed that ha and na genes of all studied aiv h n grouped in the g sub-lineage of the eurasian lineage. the highest phylogenetic relationship and nucleotide identity were shown with the prototype a/ quail/hong kong/g / rather than other aiv h n prototypes such as a/duck/hong kong/y / and a/ chicken/hong kong/g / . all algerian strains were also related to each other (nucleotides identity ranging from . to % and from . to % for the ha and na, respectively) and grouped together in the phylogenic tree (bootstrap value: for the ha and na,) (fig. ) . besides, the isolate a/chicken/algeria/ bbd/ exhibited the lowest nucleotide identity, as compared to all studied h n strains. our strains were also monophyletic with recently published algerians aiv h n sequences, such as a/chicken/algeria/ / and a/chicken/algeria/ / , isolated in the central region of algeria in ( fig. ) with high nucleotide sequence identities ( % for ha and to % for na). the algerian h n studied clustered with aiv h n from different geographic areas such as morocco (more than % genetic identity) and the united arab emirates (uae) (more than % identity), belonging to the g middle eastern group. our isolates were also highly similar to aiv h n isolated, in in burkina faso (more than % identity for both ha and na). on the other hand, the current algerian aiv h n were relatively distant from the tunisian ( % for the ha and na) and the libyan strains ( % nt identity for ha) isolated in and , respectively. as for surface genes, all internal genes (pb , pb , pa, np, m and ns) of the studied h n isolates were compared to their counterparts from genbank to establish phylogenetic trees and genetic similarity matrixes at the nucleotide level. here again, the internal genes of the algerian strains clustered within the g sub-lineage, and were closely related to each other, as shown both by their high nucleotide identities ( . - . %, . - %, . - %, . %- , . - . %, and . - % for pb , pb , pa, np, m, and ns gene segments, respectively) and the bootstrap values ( fig. ) . no reassortment event was observed. the strain a/chicken/ bdd/algeria/ remained the most distinct strain. in addition, the studied strains showed high nucleotide sequence identity with those isolated during the same year ( ), with more than , to %, to %, more than , , and % identity for pb , pb , pa, np, m, and ns, respectively. as compared to aiv h n isolated in other countries, the highest nucleotide identities and phylogenic relationship were shared with those reported in morocco, burkina faso and uae, already grouped within the g sub-lineage ( fig. ). the results of the nucleotide sequence comparison (percent identity) between the genes segments (pb , pb , pa, ha, np, na, m and ns) of the studied algerian h n strains and selective isolates are illustrated in table . given the highest nucleotide identity found between the studied isolates as well as compared to selective used strains, only three isolates, a/chicken/ bdd/algeria/ , a/chicken/ bdd/algeria/ and the most distinct a/chicken/ bdd/algeria/ isolate were presented in table . molecular markers known in the literature for their roles in pathogenesis, host tropism, enhanced replication or drug resistance have been carefully studied and are summarized in tables , , and (for ha, na, and main internal proteins). of interest, the current algerians aiv h n share a monobasic cleavage site motif rssr/ glf (h numbering) (table ) , which is similar to that of the prototype a/quail/hong kong/g / strain, defined as lpaiv in poultry. our viruses harbor ha markers of human-like α , sialic acids binding preference (a t, q l and q i). in addition, a new substitution (t a), with unknown biological function and no previous report for h n viruses, was revealed in three out the algerian strains. all studied isolates shared seven potential glycosylation sites (pgs) at the same position: - (nts), - (ngt), - (nvt), - (nst), - (nis), - (ngt) and - (ngs). the pb -f protein of algerian isolates was truncated at aa . additionally, all algerian strains harbored the s n substitution in m protein, which is associated with drug resistance. none of the known neuraminidase inhibitors resistance markers was detected in na proteins. the ns protein showed the "gsev" pdz ligand (pl) c-terminal motif and no - deletion. aiv h n has a widespread distribution. in north africa, the virus was detected in tunisia [ ] and recently in morocco [ ] . however, no official report is available on the circulation of h n viruses in algeria, except for the recent declaration of one case in the north center of the country [ ] . besides, in the province of batna (northeast algeria), an outbreak of highly contagious respiratory disease was reported in broiler flocks, in and we showed here that the outbreak was due to h n aiv. a high mortality rate ( to %) was recorded in unvaccinated broilers; such high mortality rate contrasted with a low pathogenic nature of isolated h n subtype and were certainly due to co-infections of h n aiv with other respiratory pathogens particularly infectious bronchitis virus (ibv), escherichia coli, hpaiv, mycoplasma gallisepticum (ms), ornithabacterium rhinotracheale, avian metapneumoviruses (ampv) and newcastle diseases virus (ndv) [ , [ ] [ ] [ ] [ ] . the full-length genome sequencing of aiv viruses allowed molecular characterization and more knowledge about the molecular genetic markers of virulence and pathogenicity. phylogenetic analyses of ha and na surface glycoproteins of the currents h n isolated strains showed a same evolutionary pattern. all algerians h and n gene sequences were monophyletic and grouped with different aiv h n strains from north africa and the middle east, in the g -like aiv's group. these results are not surprising given the large spread of the g -like group in the middle east, which has facilitated transmission of the disease in north africa and neighboring countries. different countries sharing borders with algeria were also infected by the same lineage, such as morocco [ ] , tunisia [ ] and libya [ ] . in the middle east, egypt was also infected with aiv h n of the g lineage [ ] . the monophyletic aspect shared with aiv h n viruses from morocco, united arab emirates, burkina faso and saudi arabia suggests virus spread in the region. the high nucleotide identities and the phylogenetic relationship between the algerian and the moroccan aiv h n isolates were not surprising and suggest that these isolates have a common origin [ ] . the reinforcement of biosecurity measures and the study of the factors of introduction and propagation of aiv h n in industrial breeding farms are crucial to reduce both the prevalence of the disease and the high economic losses. on the other hand, the low nucleotide sequence identity observed between the algerian and the tunisian strains, isolated in and - , respectively, showed that temporal distance plays a role on virus evolution here, and suggests a surveillance gap in the - period in the region. phylogenetic analyses also showed that all internal genes (pb , pb , pa, np, m, and ns) of the algerians h n strains are homogeneous and grouped with those from neighboring countries (morocco and tunisia) and more globally from the middle east, under the g lineage. despite the reassortment observed among different aiv subtypes (h , h , h ) [ ] and lineages, our findings suggest that the algerians h n viruses did not undergo genetic reassortment with other aiv subtypes and lineages. the algerians strains shared the rssr/glf (h numbering) sequence motif at their ha -ha connecting peptides, which indicates their low pathogenicity [ ] . it has been reported that this motif is characteristic of lpaiv h n viruses from middle east [ , ] and asia [ ] and well adapted to poultry [ ] . however, some studies have reported that h n viruses may undergo punctual substitutions at the ha /ha connecting peptide sequence and acquire the dibasic pattern k-s-s-r at the cleavage site [ ] , thus increasing their pathogenicity [ , ] . the aa residues / , / , / , / and / (h /h numbering) at the rbs of the ha protein are key determinants [ , ] . residue position is the most crucial for host specificity. thereby, aiv having the signature q , preferentially bind avian host receptor via α [ , ] gal-type binding [ ] . the current algerian strains harbored two aa substitutions, n h, q l, within the rbs, which suggests they may be able to infect other species than birds, such as mammalian hosts. particularly, the substitution q l enhancing the affinity shift from avian toward human-like receptors [ ] . in addition, matrosovich et al., ( ) suggested that the residue l is typical of human pandemic aiv h and h subtypes, and is not characteristic of aiv [ ] . similarly, the residue h was associated with a binding preference for human receptors on the respiratory epithelial cells [ ] , direct contact transmission of aiv h n in ferrets [ ] and more efficient replication in human airway cell cultures [ ] . two additional substitutions, a t and q i, on the rbs, were observed in the algerian h n strains. the first substitution is also present in a/duck/hong kong/y / , the prototype of y -like viruses, and a/guangdong/mz / , a human h n virus. their functions remain unknown [ , , ] ; human h n viruses; dash indicates that the aa is similar to that in the strain a/quail/hong kong/g / at the same position [ ] . at the same positions, different substitutions were previously described, such as t/v/a (h numbering) [ ] . therefore, t may allow binding of the algerian strains to α [ - ] human-like receptors, but with lower affinity as compared to residues a and v at the same position, v characteristic of hpaiv, conferring the most affinity binding towards human receptors [ ] . considering the combination of multiple substitutions in the rbs, the algerian strains may have higher binding affinity to human-like receptors. the blast analysis (ncbi) of the ha protein showed an exclusive mutation t a for our strains in comparison with h n subtype viruses published in genbank; the strains isolated the same year in the center of algeria, did not harbor this mutation. interestingly, this mutation was conserved in all human h n strains of - , in india [ ] and in italian strains [ ] . the only h n strain that displays a substitution at the same position (t d) was isolated from a -year-old child in changsha city, in china [ ] . biological impact of such mutation remains unclear and should consequently be deeply studied. another mutation, t a, without any known role in virus infectivity or host tropism, distinguished our strains; such substitution was present only in the korean-like h n viruses but absent in the moroccan strains isolated in . all the current algerian strains shared the same glycosylation pattern with seven out of eight pgs, as compared to the g -like prototype (q/hk/g / ). it has been reported that variations in pgs patterns of influenza virus may influence the pathogenicity, the affinity and the receptor binding specificity [ , ] and possibly infection of a new host. matrosovich et al., ( ) suggested that glycosylation patterns have a crucial role in the adaptation of avian influenza virus to poultry [ ] . besides, reading et al., ( ) and sun et al., ( ) have reported that a loss of pgs is associated with increased affinity of h n and h n subtypes to human-type receptors and with virulence in mice, respectively [ , ] . in contrast, additional glycosylation sites on the ha protein were shown to decrease virulence [ ] . the hb site of the na protein contains six aa that interact directly with sialic acids; this includes three residues s at positions - , n, w and k [ ] . therefore, a change in these residues would alter sialic acid binding and host receptor specificity. the current algerian isolates shared six substitutions in the na hb site: s l and s a in the first loop, s n, i n and r w in the second loop and q r in third loop, as compared to a/quail/hong kong/g / prototype; the latter showing the sequences ikkdsr sg , dsdirs and pqe in the three loops, respectively. the two substitutions (s a and r w) enhanced the cross of the species barrier and the adaptation to a mammalian host. these substitutions were also detected in h n viruses isolated in asia and the middle east, and especially in h n and h n subtypes that caused human pandemics [ , ] . these observations corroborate with the change in the binding specificity of ha protein of the algerian strains to mammalian hosts. finally, the substitution i n, in the second loop of na, offered one additional pgs to the algerian strains ( nws). the algerians h n viruses showed additional pgs in the na protein, as compared to g -like virus a/quail/ hong kong/g / ( - instead of pgs). the pgs at position , characteristics of h n viruses, was found in the algerian isolates [ ] . these observations showed that our strains may have developed an altered glycosylation pattern, which could consequently affect their pathogenicity [ ] and increase their virulence [ ] following alteration of the antigenicity or the sialidase activity [ ] and the escape from the immune defenses [ ] . likewise, the increase in the glycosylation of na protein of highly pathogenic strains promoted the cleavage of na protein by cellular proteases and consequently the spread of the infection [ ] . the na inhibitors can link different aa residues at specific positions, in the enzyme active site, including at positions , , , , , , and . it has been reported that mutations e g, r k, h y, or r k at the active site of na, allowed for the development of drug resistance [ , , ] . all algerians strains are however predicted to be sensitive to neuraminidase inhibitors based on their na sequences. although mutations in the ha gene promote infection of novel hosts and mammalian adaptation, different studies have shown that internal proteins are also important and may affect host tropism and pathogenicity [ , ] . mutations within the replication complex genes (pb , pb , and/or pa) may induce enhanced virus replication [ ] . the pa gene is considered as an important determinant in the virus adaptation to new hosts [ ] , and mutations on pa may increase virulence in animal models [ ] . the rnp complex also form host-specific genetic lineage and signatures. in addition, the m protein (m ) interacts with the vrnp, and may determine the host range [ ] . the pb protein in particular harbors different aa signatures associated with host range and efficient multiplication of aiv in mammals [ ] ; but, the most important is the aa at position . it is common that residues v, r, k and n at positions , , and , respectively, are associated with increased virulence and adaptation to mammalian host. algerian h n strains harbored residues a, q, e and d, which are associated with avian preferences [ ] [ ] [ ] . in addition, the pb residues e g [ ] , t a [ ] , k t [ ] , and t i [ ] , which may lead to increased viral replication rates and pathogenicity in mammals, have not been detected in our strains. in fact, these latter shared e, t and k. however, the residue k reported in the tunisian h n and ck/hk/ / (h n ) strains and considered as avian virus-like aa [ ] , was substituted by q in the algerian strains, with unknown function. besides, the substitution k r was also detected but was not associated with human associated h viruses [ ] . while the substitution k that confers high pathogenicity, virulence and increased replication in mice [ ] , was not detected in our algerian viruses, three substitutions r, s and t, associated with mammalian adaptation, were observed [ , ] . besides, the substitution v that contributes to an increased polymerase activity in mouse cells was observed [ ] . additionally, the algerian strains shared the substitutions v and v that are reported in the moroccan and the egyptian strains and associated with enhanced polymerase activity [ ] . molecular markers that were previously associated with avirulent strains such as v [ ] , d and f, [ ] were also observed in all algerian studied strains (table ). due to the many changes in the aa of pb of the studied strains, functional interactions between pb and other polymerase components might be affected [ ] . like adaptive mutations in polymerase activity of h n pdm and h n viruses, increased significantly the activity of h n in mammalian hosts [ ] . it seems that combined mutations may increase the activity of h n polymerase. the pb substitutions n s, k e/m and q, known to be associated with increased polymerase activity, h n pathogenicity in mice as well as adaptation to mammalians [ , , ] , were not observed in the currently circulating algerian strains, which however shared n, k and k. nevertheless, one molecular determinant (pb p) of host specificity, adaptation to mice [ , ] and enhanced polymerase activity [ ] , was detected. similarly, one other substitution d g, associated with increased polymerase activity and aiv h n virulence in mice, has also been detected [ ] . in addition, three substitutions r k (polymerase activity in mammalian cells), h y (polymerase activity and virulence in mallards, ferrets and mice) and m t (virulence related to mutation) [ ] , associated with high pathogenicity of h n , were also identified in algerian sequences. the influenza virus pa protein contain several aa substitutions that affect host range, polymerase activity and pathogenicity [ , ] . some aa, at crucial positions, may influence these functions and increase the risk for human infections [ , ] . algerian strains harbored three pa substitutions v i, k r and s n, associated with adaptation to mammals [ , , ] (table ). the unique aa substitutions such as v i, t i, k n/e, s i and r k, which increase aiv polymerase activity, replication in mammalian cells and virus virulence in mice, were however not detected [ , [ ] [ ] [ ] . among the aa associated with increased virulence, pa protein of current algerian strains shared three, v, l and l, different molecular markers of virulence [ , , ] . likewise, the substitution t a that was shown to reduce lethality of hpaiv h n in avian species, polymerase activity in mammalian cells and virulence in duck [ ] , was absent. instead, all algerians strains exhibited the t residue, associated with enhanced lethality. the studied strains retained the mutation d, which is highly conserved in avian influenza viruses and g- reference strain. song et al., ( ) reported that this residue increases polymerase activity in mammalian and avian cell lines and may therefore allow species barrier crossing and human cases [ ] . influenza virus nucleoproteins have different aa residues that directly interact with the arn polymerase, particularly with pb and pb sub-units [ ] . np has multiples functions in aiv biological cycle, replication/pathogenicity and mammalian host infection [ ] . different substitutions affecting np functions have previously been reported. the aa sequence analyses of the algerian h n nucleoproteins showed conserved g- lineage residues d, r, k, t, d and d, associated with avian hosts [ , , , ] . zhu et al. ( ) reported that some substitutions in np modulate the replication ability of g- virus in mammalian hosts. we also found that our isolates shared the aa asp (d) at position , which is associated with increased polymerase activity in mammalian cells, and increased replication of h n viruses especially at low temperatures [ ] . such mutations may therefore favor the replication of aiv h n in the human upper respiratory tract. the np substitution e d was previously described for a/ chicken/bangladesh/vp / (h n ) [ ] and considered as common in human associated h viruses [ , ] . m and m proteins harbored different crucial sites related to host tropism, immune response [ ] and resistance to antivirals m blockers [ ] . the m molecular determinant of virulence i, conserved among h n lineage [ ] and associated with mammalian preference [ ] , were nevertheless observed. in addition, all strains possessed two residues, d and a, as reported in m of h n isolated from human cases and known to increase virulence in mice [ ] . interestedly, the algerians strain harbored a number of single aa substitutions at positions , and , that were shown to contribute to the increase of virulence of hpaiv h n . fang et al., ( ) reported that the substitution n d and t a, increase virulence and lethality in mice [ ] . as compared to a/duck/hokkaido/wz / hpaiv h n strain, aa sequence alignments showed that all algerians strains shared the substitutions i m [ ] , reported to increase virulence in mice, chickens and ducks. the m proteins of algerian viruses retained the molecular marker l of g- lineage and residues t, s, y, q and v shown to enhance avian infections [ , ] . analyses of other molecular markers of host range and virulence showed three m molecular markers d, v and f, associated with mammalian transmission and human cases [ ] and the two molecular virulence determinants, s and p [ ] . the m protein is also considered as a target for antiviral drugs. mutations in crucial positions can promote the development of antiviral resistance phenotype. among aa substitutions associated with resistance to antiviral drugs, at positions , , , , and , current algerian strains only harbor one substitution ( n), suggesting their resistance to amantadine and rimantadine [ , ] . the ns protein of the algerian strains are typical of h n influenza virus with a pdz (x-s/t-x-v) gesv c-terminal motif, characteristic of avian species [ , ] . parvin et al., ( ) reported, however, that most aiv have esev (pdz domain) at the end of their ns protein [ ] , increasing the virus virulence in mice [ ] . our strains exhibited the ns substitution e g as compared to the g- like ( e) and the mammalian preference viruses ( k/r) [ , ] . the substitution e g may indeed modulate the pathogenicity of the studied isolates, through structural change in the pdz c-terminal motif, but their biological significance remains unknown. algerian isolates shared the ns residue s [ , ] , which was shown to increase the virulence in mice and to decrease the antiviral immune response of host cells [ ] . two additional markers m [ ] and v a [ ] , which increase viral replication in mammalian cell and virulence in mice and decrease interferon response in chicken, were detected. the present study reported, for the first time, the fulllength genome sequences of ten aiv h n , recently isolated from a fatal influenza outbreak with respiratory manifestations that occurred in northeast of algeria, in april . phylogenetic and molecular analyses were used to study the full genome of these aiv isolates. phylogenetic analysis showed that the surface and the internal genes have the same evolution patterns and are grouped with the moroccan and the middle eastern strains, inside the g -like h n . the blast analysis demonstrated that all strains are also highly similar to the algerian strains isolated during the same year, in the central region of algeria ( ). these strains were similar to aiv h n isolated in morocco ( ). the implementation of adequate biosecurity measures to limit the introduction of the infection into the algerian poultry farms is highly recommended. evolution of influenza a viruses in wild birds functional balance between neuraminidase and haemagglutinin in influenza viruses characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls new world bats harbor diverse influenza a viruses avian influenza virus infections. i. characteristics of influenza a/turkey/wisconsin/ virus a brief summary of the epidemiology and genetic relatedness of avian influenza h n virus in birds and mammals in the middle east and north africa avian influenza a (h n ): computational molecular analysis and phylogenetic characterization of viral surface proteins isolated between and from the human population prevalence of avian respiratory viruses in broiler flocks in egypt co-infection with multiple respiratory pathogens contributes to increased 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genetic conservation of hemagglutinin gene of h influenza virus in chicken population in mainland china characterization of h n avian influenza viruses from the middle east demonstrates heterogeneity at amino acid position in the hemagglutinin and potential for transmission to mammals characterization of avian h n influenza viruses from united arab emirates phylogenetic analysis of the hemagglutinin gene of recent h n avian influenza viruses isolated from broiler flocks in iran h n influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens novel genotypes of h n influenza a viruses isolated from poultry in pakistan containing ns genes similar to highly pathogenic h n and h n viruses structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid receptor specificity of influenza viruses and its alteration during interspecies transmission replication and transmission of h n influenza viruses in ferrets: evaluation of pandemic potential amino acid in the hemagglutinin of h n influenza viruses determines cell tropism and replication in human airway epithelial cells h n influenza a viruses from poultry in asia have human virus-like receptor specificity genetic and biological characterization of h n avian influenza viruses isolated in china from genetic characterization of circulating a (h n ) pdm influenza viruses from eastern india early cocirculation of different clades of influenza a/h n v pandemic virus in northern italy full-length genome analysis of an avian influenza a virus (h n ) from a human infection in changsha city structural differences among hemagglutinins of influenza a virus subtypes are reflected in their antigenic architecture: analysis of h escape mutants the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice nlinked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the pandemic and seasonal h n influenza a viruses neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza h n viruses for mice structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens avian-tohuman transmission of h n subtype influenza a viruses: relationship between h n and h n human isolates h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong molecular changes in the polymerase genes (pa and pb ) associated with high pathogenicity of h n influenza virus in mallard ducks genetically destined potentials for nlinked glycosylation of influenza virus hemagglutinin threedimensional structures of influenza virus neuraminidase-antibody complexes genomic sequences of human infection of avian-origin influenza a (h n ) virus in zhejiang province genomic signatures of human versus avian influenza a viruses amino acid changes in the influenza a virus pa protein that attenuate avian h n viruses in mammals identification of mammalian-adapting mutations in the polymerase complex of an avian h n influenza virus the viral polymerase mediates adaptation of an avian influenza virus to a mammalian host molecular basis for high virulence of hong kong h n influenza a viruses host restriction of avian influenza viruses at the level of the ribonucleoproteins molecular correlates of influenza a h n virus pathogenesis in mice transmission of influenza virus in a mammalian host is increased by pb amino acids k or e/ n 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analysis identifies functional domains in the influenza a virus pb polymerase subunit pb subunit of avian influenza virus subtype h n : a pandemic risk factor genomic and protein structural maps of adaptive evolution of human influenza a virus to increased virulence in the mouse glycine at position in pb contributes to the virulence of h n avian influenza virus in mice mutations in the pa protein of avian h n influenza viruses affect polymerase activity and mouse virulence inventory of molecular markers affecting biological characteristics of avian influenza a viruses molecular changes associated with the transmission of avian influenza a h n and h n viruses to humans role of host-specific amino acids in the pathogenicity of avian h n influenza viruses in mice molecular determinants of virulence and stability of a reporter-expressing h n influenza a virus amino acid substitutions v i or a s/i t/v i/v a in the pa n-terminal domain increase the virulence of h n influenza a virus synergistic effect of s p and n d substitutions in the pa of h n avian influenza virus contributes to mammalian adaptation influenza virus nucleoprotein interacts with influenza virus polymerase proteins the ecology and adaptive evolution of influenza a interspecies transmission. influenza respir viruses residues v and/or d in the np protein enhance polymerase activities and potential replication of novel influenza (h n ) viruses at low temperature evolution of the m gene of the influenza a virus in different host species: large-scale sequence analysis two amino acid residues in the matrix protein m contribute to the virulence difference of h n avian influenza viruses in mice a single amino acid in the m protein responsible for the different pathogenic potentials of h n highly pathogenic avian influenza virus strains structure and mechanism of the m proton channel of influenza a virus a comprehensive surveillance of adamantane resistance among human influenza a virus isolated from mainland china between a new influenza virus virulence determinant: the ns protein four c-terminal residues modulate pathogenicity persistent host markers in pandemic and h n influenza viruses a single-amino-acid substitution in the ns protein changes the pathogenicity of h n avian influenza viruses in mice a single amino acid substitution in the novel h n influenza a virus ns protein increases cpsf binding and virulence the ns gene contributes to the virulence of h n avian influenza viruses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the institute of veterinary and agronomic sciences, batna, algeria and the french ministry of agriculture for supporting this research. amino acids analysis showed that the algerians h n strains carried out different molecular markers associated with affinity to human-like receptors and increased virulence. interestingly, different substitutions, common to h n viruses and exclusive to the algerian strains, were also revealed, indicating continuous evolution of aiv in the field. therefore, further studies to monitor such strains evolution in the field and reduce the risk of virus transmission to humans are crucial. additional studies using animal models are also required to investigate the pathogenicity of current algerian h n strains. key: cord- -xfzf ath authors: lambertz, ruth lydia olga; gerhauser, ingo; nehlmeier, inga; gärtner, sabine; winkler, michael; leist, sarah rebecca; kollmus, heike; pöhlmann, stefan; schughart, klaus title: h influenza a virus is not pathogenic in tmprss knock-out mice date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: xfzf ath the host cell protease tmprss cleaves the influenza a virus (iav) hemagglutinin (ha). several reports have described resistance of tmprss (−/−) knock-out (ko) mice to iav infection but iav of the h subtype have not been examined yet. here, we demonstrate that tmprss is able to cleave h -ha in cell culture and that tmprss (−/−) mice are resistant to infection with a re-assorted pr _ha(h ) virus. infection of ko mice did not cause major body weight loss or death. furthermore, no significant increase in lung weights and no virus replication were observed in tmprss (−/−) mice. finally, only minor tissue damage and infiltration of immune cells were detected and no virus-positive cells were found in histological sections of tmprss (−/−) mice. in summary, our studies indicate that tmprss is required for h iav spread and pathogenesis in mice. these findings extend previous results pointing towards a central role of tmprss in iav infection and validate host proteases as a potential target for antiviral therapy. the hemagglutinin (ha) of influenza a virus (iav) facilitates viral entry into target cells. for this, ha binds to cellular receptors and fuses the viral membrane with a target cell membrane. ha is synthesized as an inactive precursor molecule, ha , and needs to be processed by host proteases into the subunits ha and ha in order to acquire membrane fusion competence [ ] [ ] [ ] [ ] . it has been shown previously that the transmembrane protease serine (tmprss ) is required for cleavage of ha of subtypes h , h and h in infected mice [ ] [ ] [ ] [ ] . tmprss cleaves these has at a single monobasic cleavage site [ , , ] . here, we demonstrate that also the ha from h iav subtypes requires processing by tmprss in vivo for virus replication and pathogenesis. first, we investigated the cleavage of ha from subtype iav (h -ha) by tmprss in cell culture. for this, we cotransfected t cells with plasmids encoding h -ha from a/mallard/alberta/ / (h n ) and murine tmprss [ ] . transfection of empty plasmid served as negative control while treatment of ha expressing cells with trypsin served as positive control. as shown in fig. , trypsin and tmprss cleaved ha, as determined by the detection of the ha cleavage product ha . the slight differences in the size of the ha bands produced by tmprss relative to trypsin have been documented previously and reflect differences in nglycosylation of ha [ ] . for infection studies in mice, we generated a ( + ) re-assorted virus carrying the h -ha from the a/mallard/alberta/ / (h n ) virus on the backbone of a/puerto rico/ / (h n , pr ) virus. in this way, results were independent of other gene segments from the donor bird virus and comparable to other studies in which the ha segment was exchanged and tested on an isogenic pr background [ ] . for the generation of the pr _ha(h ) virus, the ha encoding segment from the avian virus was cloned by sequence and ligation independent cloning as described earlier [ ] into plasmid phw- (kindly provided by robert webster, st. jude, memphis, usa) using primers ′-gacctcc-gaagttgggggggagcaaaagcagggg- ′ and ′-ttttgggccgccgggttattagtagaaacaagggtgtttt- ′. re-assorted virus was then rescued from plasmids as described earlier [ ] with ng of each plasmid, . μl transit- (mirus) in μl optimem (gibco) using the h encoding plasmid and plasmids containing all other seven segments of pr (kindly provided by robert webster, st. jude, memphis, usa). the resulting virus pr _ha(h ) virus was propagated in the chorioallantoic cavity of -day-old specific pathogen free (spf) embryonated chicken eggs (charles river laboratories, germany) for h at °c. virus rna was extracted, and quality and integrity were controlled using agilent technologies bioanalyzer (agilent technologies; waldbronn, germany). a sequencing library was generated from ng total rna using totalscript rna-seq kit (epicentre) without fragmentation, according to the manufacturer's protocol. the libraries were then sequenced on illumina miseq using miseq reagent kit v ( cycles, paired end runs) and the correct sequence of the re-assortant virus was confirmed. the titer of the stock viruses was determined by focus forming unit (ffu) assay [ ] . for in vivo studies, female tmprss −/− (b . s -tmprss tm tsyk) [ , ] and c bl/ jrj wild type (wt) mice (janvier, - weeks old) were infected intranasally with × ffu pr _ha(h ) as described before [ ] and changes in body weight and survival were monitored for the next days. animals with a body weight loss of more than % were euthanized and recorded as dead in addition to mice that were found dead. we did not observe dead animals nor significant body weight loss in infected tmprss −/− mice, whereas wt mice exhibited significant body weight loss and some mortality after infection with pr _ha(h ) virus (fig. a, b) . titers were then determined by ffu assay in each lung as described in [ ] . viral replication in lungs of infected (dose of × ffu) female tmprss −/− and wt mice ( - weeks old) revealed no detectable virus replication in tmprss −/− mice, whereas wt mice showed increased lung titers at day and post infection (dpi) (fig. c) . we then investigated other parameters for pathogenicity by measuring relative lung weights, tissue damage and infiltration of immune cells. wt infected mice showed a significant increase in relative lung weights (indicative of hyperemia, pulmonary edema, and/or leukocyte extravasation) compared to tmprss −/− mice that exhibited similar relative lung weights as mockinfected mice (fig. d , e). at and dpi, eye blood was collected and lymphocytes, monocytes and granulocytes were counted using the vetscan hm hematologic system as described previously [ ] . the analysis of blood cells showed a significant increase of granulocytes in wt mice from to dpi compared to mock infected mice, whereas tmprss −/− mice only showed a marginal increase in granulocytes compared to mock-infected animals (fig. e ). an increase of granulocytes is an indicator for the severity of the infection [ ] . we then performed histopathological studies on tissue sections from pr _ha(h ) infected tmprss −/− and wt mice (fig. ) . female -week-old wt and tmprss −/− mice were infected intranasally with × ffu pr _ha(h ). lungs were prepared at dpi and paraffin sections were stained with hematoxylin and eosin (h&e). infection was associated with severe bronchiolar epithelial damage and moderate inflammatory cell infiltration (fig. a , b) in wt mice, whereas lungs of tmprss −/− mice exhibited only mild or no damage of bronchiolar epithelium (fig. a, b) . infiltration of mononuclear cells including lymphocytes and macrophages into interstitium and alveolar lumina adjacent to bronchioli was more pronounced in wt compared to tmprss −/− mice (fig. a, c) . for immunohistochemical detection of infected cells, a primary antibody against iav nucleoprotein (ebs-i- , european veterinary laboratory) and a secondary hrp-labeled goat-anti-mouse igg a (southern biotech) were used [ ] . analysis was performed by counting all iav-infected cells in randomly selected high power fields ( × . mm ) on one immunohistochemically stained section of the complete lung per animal. iav antigen was detected in bronchiolar epithelial cells and leukocytes of wt mice, whereas in lungs of tmprss −/− mice no antigen positive cells were observed (fig. a, d) . this finding corroborates lack of virus detection by ffu titration in lung homogenates at and dpi. together, these observations showed that tmprss −/− mice were resistant to pr _ha(h ) virus-induced disease. we then analyzed cleavage of viral ha from bronchoalveolar lavage (bal) isolates. whole lung extracts will contain cleaved and non-cleaved ha that is being produced inside cells. in bal secretion, mainly viral particles are detected that are released from infected cells. therefore, bal is optimal source to test presence of the hemagglutinin and its cleavage state. total protein was determined in bal samples using the pierce bca protein assay kit according to the manufacturer's instructions and μg protein was run on a . a mean body weight in percent of starting weight ± standard error of mean (sem). b survival curve. statistics for survival curve was calculated with the log rank test. ns: non-significant. c viral load in lung at (wt: n = ; ko: n = ) and dpi (wt: n = ; ko: n = ). mean titers are shown ± sem. the detection limit for the ffu assay was . undetectable titers were set to . d mean (grey) ± sem of the relative lung weight (lung weight* /body weight) determined on (wt: n = ; ko: n = ; mock (m ): n = ) and dpi (wt: n = ; ko: n = ). e on dpi (wt d : n = ; ko d : n = ; mock m d : n = ) and dpi (wt d : n = ; ko d : n = ), eye blood was collected. cell types (lymphocytes, monocytes and granulocytes) were phenotyped and counted based on cell volume using the vetscan hm hematologic system. cell types are presented in percentage of total white blood cells (wbc). mock samples were collected from wt mice. to note, we did not compare basal cell counts in ko versus wt since a difference is not expected, although it cannot be excluded. bars represent mean values, error bars show ± sem. statistics for (c) and (d) were calculated by mann whitney tests (** p < . ; *** p < . ) sds-page, blotted on a pvdf-membrane (bio-rad), which was then incubated with an anti-h n polyclonal antibody (sino biological, , -rp , hb de -b) using a dilution of : in tbst with . % milk powder and then with an horseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody (sigma, a ) in a dilution of : , . as a reference (internal control) for total viral particles in a sample, we used the viral np protein. if such a signal is present, viral particles have been produced and viral proteins can be detetcted. detection of signals was performed using the fujifilm las- imaging system. subsequently, the membrane was incubated with an anti-np antibody (genetex, gtx ) in a dilution of : , , followed by and leukocytes (arrowhead) were found in wt mice, whereas no immunostaining was detectable in tmprss −/− mice (see inserts). b necrotic bronchioli were determined as percentage of total cells. c semi quantitative scoring results of cellular infiltration ( = none, = mild, = moderate, = severe). d iav antigen positive cells were counted twice in randomly selected high power fields ( × . mm ). bars indicate mean values +/− sem. one-sample t-tests were used for statistical analysis (* p < . ; ** p < . ). please note that all values are identical for wt in the inflammation score (error bar is zero) incubation with the same secondary antibody as before. in the bal of infected wt mice, both ha and ha were detectable (fig. ) . also, a very faint ha band was detectable in tmprss −/− mice after image enhancement (fig. b) . the np band was much less intense in tmprss −/− mice than in wt mice and only clearly detectable after image enhancement (fig. a, b) . nevertheless, the presence of np demonstrated that viral particles were present in the samples from tmprss −/− mice that ha was detected but no cleaved ha . of note, a band of the size of ha was observed in bal of tmprss −/− mice that was also detectable in mock infected lung at approximately the same intensity. thus, this band in tmprss −/− mice is unspecific and not representing ha . taken together, these results are consistent with the observed absence of pr _ha(h ) (h n ) replication in tmprss −/− mice. since no viral replication and only minor immune responses were detected in the studies described above, we confirmed that mice had indeed been infected with the pr _ha(h ) virus by determining the presence of h -specific antibodies at dpi in the serum. for this, female mice were infected with × ffu pr _ha(h ). at dpi, sera from infected and pbs mock-treated mice were prepared and analyzed for the presence of iav-specific antibodies. -well plates were coated with × ffu pr _ha(h ) viruses ml − , diluted in x pbs (phosphate buffered saline), incubated overnight, washed with pbst ( x pbs, . % tween ), and blocked with pbst-fcs (pbst, % v/v fetal calf serum). fifty μl of : diluted mouse serum was added and incubated for hours (h) at °c. plates were then washed three times with pbst and μl of anti-mouse igg (goat anti-mouse igg heavy chain gamma-hrp, kpl # - ), diluted : in pbst-fcs, were added and incubated for h at °c. wells were then washed three times with pbst and μl of substrate (sureblue tmb peroxidase substrate, kpl, # - - ) was added. after minutes, the reaction was stopped by adding μl tmb stop solution (kpl, # - - ) and the optical density was measured at nm. our studies showed that infected tmprss −/− mice exhibited a significant seroconversion that was similar to infected wt mice (fig. ) . thus, incoming virus was able to infect lung cells and trigger an immune response but was not able to spread and replicate to levels detectable in ffu assays. it may be worth noting that in mouse lungs, tmprss gene expression is down-regulated at the transcriptional level after infection [ ] . we showed that tmprss −/− mice were resistant to h iav pathogenesis. after infection with pr _ha(h ) tmprss −/− mice did not lose body weight and no viral replication was observed in lungs at and dpi. histopathological analysis showed strongly reduced inflammatory lesions and the lack of detectable viral antigen in tmprss −/− mice. we and others demonstrated earlier that tmprss −/− mice are also resistant to h iav infection. in these studies, infectious virus was detected at day two after infection in lung homogenates [ , , ] . thus, some residual replication was observed for h virus, whereas in our present study, pr _ha(h ) virus was not able to replicate to detectable titers in tmprss −/− mice. this difference in detectable virus titers might be due to the origin of the two viruses. the h -ha viruses (a/puerto rico/ / or a/california/ / fig. the h -ha is not processed in tmprss −/− mice. female - -week-old c bl/ j wild type (wt) and tmprss −/− knock-out (ko) mice were infected intranasally with × focus forming units (ffu) pr _ha(h ). on day post infection (dpi) broncho-alveolar lavages (bal) were prepared. a h cleavage and nuclear protein (np) in bal (day p.i.) were analysed by western blots. b enhanced image of (a) using imagej software (b/c: brightness/contrast). ha : uncleaved ha, ha : n-terminal part of cleaved ha, ha : c-terminal part of cleaved ha; np: nuclear protein ) were adapted to mouse [ , ] , whereas the ha encoding segment of the h -ha virus in our study originated from a bird isolate. it is well conceivable that the bird h -ha is missing adaptations that have been acquired by the mammalian h -ha and allow for more efficient replication in tmprss −/− mice. the very low amount of virus particles in tmprss −/− mice precluded the formal demonstration that absence of viral spread and pathogenesis in these animals was due to lack of ha activation. however, several observations support this interpretation: only a weak np signal and no ha signal were detected in samples from tmprss −/− animals, even upon overexposure of the immunoblot. moreover, our studies in cell cultures showed that ha can be cleaved by tmprss and there is at present no evidence that tmprss can promote iav spread by other means than ha cleavage. these points strongly suggest that the absence of proteolytic processing of ha is the underlying cause for the lack of replication and spread of h -ha iav in tmprss −/− mice. in conclusion, our study extends the list of iav subtypes known to depend on tmprss for viral spread in mice to the h subtype. the present and previous results identify tmprss as an attractive target for novel anti-iav drugs. the hemagglutinin: a determinant of pathogenicity hemagglutinin activating host cell proteases provide promising drug targets for the treatment of influenza a and b virus infections influenza virus activating host proteases: identification, localization and inhibitors as potential therapeutics influenza virus structural and nonstructural proteins in infected cells and their plasma membranes tmprss is essential for influenza h n virus pathogenesis in mice the host protease tmprss plays a major role in in vivo replication of emerging h n and seasonal influenza viruses tmprss is a host factor that is essential for pneumotropism and pathogenicity of h n influenza a virus in mice tmprss knock-out mice are resistant to h influenza a virus pathogenesis activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium influenza and sars-coronavirus activating proteases tmprss and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts tmprss and tmprss facilitate trypsinindependent spread of influenza virus in caco- cells exchange of amino acids in the h -haemagglutinin to h residues is required for efficient influenza a virus replication and pathology in tmprss knock-out mice universal primer set for the full-length amplification of all influenza a viruses phenotypic analysis of mice lacking the tmprss -encoded protease cellular changes in blood indicate severe respiratory disease during influenza infections in mice rnaseq expression analysis of resistant and susceptible mice after influenza a virus infection identifies novel genes associated with virus replication and important for host resistance to infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions ks, rlol, ig and sp conceived and designed the study. rlol, ig, hk, srl, sg, mw and in performed the experimental analyses. rlol, sp, ig and ks analyzed the data. ks and rlol wrote the manuscript. all authors read and approved the final version. this work was supported by intra-mural grants from the helmholtz-association (program infection and immunity) and a start-up grant from the university of tennessee health science center. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. all data presented in this manuscript is included in the text. all experiments in mice were approved by an external committee according to the national guidelines of the animal welfare law in germany (bgbl. i s. , and bgbl. i s. ). the protocol used in these experiments has been reviewed by an ethics committee and approved by the relevant authority, the 'niedersächsisches landesamt für verbraucherschutz und lebensmittelsicherheit, oldenburg, germany' (permit numbers: . - - / , . . - - / ; . - , - - / ). not applicable. the authors declare that they have no competing interests. key: cord- -yvid qps authors: bisimwa, patrick n.; ongus, juliette r.; tiambo, christian k.; machuka, eunice m.; bisimwa, espoir b.; steinaa, lucilla; pelle, roger title: first detection of african swine fever (asf) virus genotype x and serogroup in symptomatic pigs in the democratic republic of congo date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: yvid qps background: african swine fever (asf) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. the analysis of variable regions of african swine fever virus (asfv) genome led to more genotypic and serotypic information about circulating strains. the present study aimed at investigating the genetic diversity of asfv strains in symptomatic pigs in south kivu province of the democratic republic of congo (drc). materials and methods: blood samples collected from asf symptomatic domestic pigs in of districts in south kivu were screened for the presence of asfv, using a vp gene-specific polymerase chain reaction (pcr) with the universal primer set ppa -ppa . to genotype the strains, we sequenced and compared the nucleotide sequences of ppa-positive samples at three loci: the c-terminus of b l gene encoding the p protein, the e l gene encoding the p protein, and the central hypervariable region (cvr) of the b l gene encoding the j l protein. in addition, to serotype and discriminate between closely related strains, the ep l (cd v) gene and the intergenic region between the i r and i l genes were analyzed. results: asfv was confirmed in of pigs tested. however, only and ppa-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p (b l) and p (e l). all the asfv studied were of genotype x. the cvr tetrameric repeat clustered the asfv strains in two subgroups: the uvira subgroup ( trs repeats, aaaabnaaba) and another subgroup from all other strains ( trs repeats, aabnaaba). the phylogenetic analysis of the ep l gene clustered all the strains into cd v serogroup . analyzing the intergenic region between i r and i l genes revealed that the strains were identical but contained a deletion of a -nucleotide internal repeat sequence compared to asfv strain kenya . conclusion: asfv genotype x and serogroup was identified in the asf disease outbreaks in south kivu province of drc in – . this represents the first report of asfv genotype x in drc. cvr tetrameric repeat sequences clustered the asfv strains studied in two subgroups. our finding emphasizes the need for improved coordination of the control of asf. pigs are increasingly contributing to improved nutrition and household incomes in regions of africa where pork consumption and pig keeping are culturally acceptable [ ] . despite the importance of pig farming, this sector is facing several constraints, with infectious disease burden being the major problem [ ] . african swine fever virus (asfv) causes an acute, highly contagious, and fatal disease in domestic pigs, with clinical signs such as fever and haemorrhagic lesions [ ] . there are currently no vaccines available to combat african swine fever (asf). the first recorded asf outbreaks were reported in pigs belonging to european settlers in kenya in [ ] . the disease continues to spread throughout eastern europe since [ ] and was reported in belgium and china in [ ] [ ] [ ] . asfv is a large, enveloped, doublestranded dna arbovirus belonging to the genus asfivirus, and the only member of the family asfarviridae [ , , ] . warthogs, bush pigs and the soft tick of the genus ornithodoros are the major reservoirs of asfv [ ] . the contagious nature and the ability to spread rapidly in pig populations over long distances makes it the most feared disease of domestic pigs [ , ] . the genome size varies from to kbp and encodes between and open reading frames, depending on the virus strains [ ] . to date, asfv genotypes have been reported worldwide based on the b l gene, which encodes the capsid protein p , and all of them are known to circulate in africa [ ] [ ] [ ] . using the serotype-identification approach [ ] , an additional asfv serotypes have been reported based on the ep r gene encoding the cd v protein [ ] [ ] [ ] . distinct antigenic types of asfv were identified based on haemadsorption inhibition (hai) serological typing, where asf protective immunity was shown to be serotype-specific, and viruses belonging to identical serogroups cross-protected against each other [ ] . this has significant importance for vaccine development. the cd v protein, encoded by the asfv ep r gene, is a transmembrane glycoprotein located in the viroplasm (around viral factories) and in the plasma membrane of infected cells. it is among the most variable genes in the asfv genome [ , ] . haemadsorption involves adhesion of pig erythrocytes to the surface of asfv infected cells, a key requirement is expression of cd v in asfv-infected cells [ ] . control of the disease is relying on surveillance, restriction of pigs and pork products movement, and rapid diagnosis and culling of asfv infected animals. the implementation of these measures is particularly difficult for african pig farmers of which many can be characterized as smallholders, due to limited capacity and appropriate policy. in , asf outbreaks were reported in more than african countries with the highest number of outbreaks ( ) registered in the democratic republic of congo (drc) causing a loss of , pigs [ , ] . previous studies have reported circulation of genotype i, ix and xiv in drc, encouraging the need for continued characterization of asfv strains responsible for outbreaks to better understand the spread of this economical important disease in drc. several variable regions of the asfv genome have been extensively used as targets for molecular epidemiology studies of asfv strains [ ] [ ] [ ] . however, previous studies achieved high resolution for discrimination between different virus strains when combining p , p and b l (central variable region or cvr) proteins [ , ] . moreover, the ep r gene encoding the cd v protein and the intergenic region between the i r and i l have also been used to characterize asfv from various locations and to track virus spread [ , ] . the first report of the presence of asf in drc was in [ ] . south kivu province is an area in the eastern drc where suspected cases of asf appear regular. reports from the provincial ministry of agriculture livestock and fishery (pmalf) and the local veterinary body indicated the death of pigs out of that presented clinical signs of asf in (report of the pmalf, unpublished data). more recently, we have used a combination of p and p proteins to characterize asfv genotype ix in apparently healthy pigs in south kivu province sampled in [ ] . it was the first study of asfv in the south kivu province. however, despite report of frequent incidences of asf in the south kivu province by the pmalf, information about asfv strains in circulation in suspected infected animals is lacking. therefore, the present study was set up to identify and characterize asfv strains in infected domestic pigs with symptoms of asf from different smallholder farms in the south kivu province in order to increase epidemiological knowledge of asfv, and to generate information for improvement of control strategies. ethical approval for the study reported here and the permission for the collection of samples was provided by the interdisciplinary centre for ethical research (cire) established by the evangelical university in africa, bukavu, dr congo, with reference (uea/sgac/km / ). a consent form which described the aim of the study was signed by farmers willing to participate in the study after translation into local languages. the study was carried out in south kivu province of the democratic republic of congo (drc), situated in the eastern part of the country. it is a large region with an area of , km , located between longitudes ° ′ ″ and ° ′ east, latitudes ′ ″ and ° ′ ″ south. comparatively, the south kivu province is over two times the size of burundi ( , km ) and rwanda ( , km ) put together. the province experiences two main seasons: a -month long rainy period, from september to may, and a -month dry period (june to august). the annual average rainfall is mm. six out of eight territories were selected for purposes of this study including; fizi, kabare, kalehe, mwenga, uvira and walungu (fig. ) . a key factor in selecting the sample sites was the inclusion of the main pig-producing, marketing, and consuming areas, with a particular focus on locations where suspected asf outbreaks had been recently reported by the provincial ministry of agriculture livestock and fishery. a cross-sectional study was conducted where the target population was households that keep pigs, and where suspected asf cases were notified based on the reports from the local veterinary and provincial ministry of agriculture livestock and fishery during december to january . in general, the number of pigs per farm varied between and and they shared housing. thus, farms with pigs presenting symptoms such as high fever, reddening of the skin, particularly ears and snout, coughing and difficulties in breathing, hemorrhagic diarrhea and vomiting, inability to walk, loss of appetites, general weakness, were considered for sampling and all the pigs were sampled. drc has a pig population of approximately million. the recommended sample size for a population of that size, using a confidence level of % and a margin of error of % would be [ ] . based on this, a total of blood samples from suspected asfv infected pigs were collected in edta (anticoagulant) tubes and were used for pcr analysis. all blood samples were collected from the jugular vein of pigs of over months of age. after collection, all collected blood samples were transported to the université evangélique en afrique (uea) and stored at − °c before being shipped on ice packs to the pan african university institute of science technology and innovation (pauisti) in nairobi then to beca-ilri hub, for subsequent analysis. total dna was extracted directly from μl of whole blood using the dneasy blood and tissue kit (qiagen, usa) following the manufacturer's recommendations. to detect the presence of asfv dna, polymerase chain reaction (pcr) amplification assay was carried out using the asfv diagnosis primers ppa /ppa (peste porcine africaine) that target the virus vp (p ) coding region to generate an amplicon of bp [ ] . pcr products were confirmed using a % agarose gel electrophoresis. all ppa-positive samples were characterized in subsequent analyses. five separate pcr experiments were set up to amplify ppa-positive samples: (i) for p genotype classification, the c-terminal region of the p protein gene was amplified using the primers p -u/d [ ] ; (ii) for p genotyping, the gene e l encoding the p protein was amplified using the primers ppa /ppa [ ] ; (iii) the b l gene characterized by the central variable region (cvr) was amplified using the primer pairs cvr-fl and cvr-fl as previously reported [ ] ; (iv) for determining the origin, and to distinguish between closely related asfv strains circulating in the south kivu province, a bp fragment, specific for identification of tandem repeat sequences (trs), located between the i r and i l genes was amplified using the primer pairs eco a and eco b [ ] ; (v) to determine the serogroups of the strains, the partial ep r gene encoding the cd v protein [ ] was amplified and sequenced using two sets of primers to generate two overlapping fragments. the primers used for the diagnosis and genotyping are presented in the table . pcr amplicons were confirmed using % agarose gel electrophoresis in the presence of molecular weight markers. for sequencing, pcr products were purified using quick pcr purification kit (qiagen, usa) following the manufacturer's instructions and sent to macrogen europe bv (amsterdam, the netherlands) for sanger sequencing. open reading frames present within the sequences generated from the amplified cvr dna fragments were translated into amino acid sequences using emboss-transeq software [ ] . both strands of purified amplicons were sequenced using the primer sets for genotyping described above. to verify similarity with known sequences, the amplicon sequences obtained were submitted to blast (basic local alignment search tool) [ ] against non-redundant genbank database. multiple sequence alignments of sequences were generated using clustal w [ ] , whereas for each locus, the unrooted maximum likelihood method (ml) phylogenetic tree with bootstrap replications was estimated by mega program and kimura -parameter model [ ] was selected based on the best-fit substitution model (ml) with the lowest bayesian information criterion (bic) score. asfv sequence data of strains and isolates available in the genbank were included as references. sequences from this study have been submitted to genbank with accession numbers mn to mn (for p ) and mn to mn (for p ). a total of blood samples collected from symptomatic pigs were screened for the presence of the asf viral dna using conventional pcr with the diagnostic primers ppa /ppa . a total of blood samples showed clear amplicons of the expected size ( bp) and data were distributed as shown in table , with the highest number of positive samples found in the uvira territory / ( . %), while the lowest was in mwenga / ( . %). sequence analysis of asfv based on the b l (p ), e l (p ) and b l (cvr) genes of the asfv positive samples using ppa diagnostic primers, we successfully amplified and sequenced ( . %) samples for p and ( . %) samples for p ( table ). the p amplicon sequences shared - % identity due to some few synonymous mutations while the p sequences were % identical (data not shown). thus, clustal protein sequence alignment showed % identity between all the p and p sequences in the study samples. sequences of p and p amplicon were compared with other p and p asfv sequences retrieved from the gen-bank database and the phylogenetic analysis revealed that the south kivu asf virus strains analyzed clustered with p genotype x including strains reported in previous studies in burundi (af ), kenya (ay ) and tanzania (jx , af , mf ) ( fig. a and b) . this is the first report of genotype x in the drc. in addition, the predicted amino acid sequences of the cvr nucleotide sequences were generated from samples ( fizi, kabare, kalehe, mwenga, uvira, walungu) and specific features based on the previously reported asfv tetrameric amino acid repeats within the cvr [ , ] were obtained. analysis of the cvr signature of the b l gene showed two different signatures when compared with sequences of strains of the same genotype from burundi, tanzania and uganda. all asfv strains contained a cvr with tetrameric amino acids, namely cast (a), cadt (b) and nvdt (n). however, in strains from the uvira territory the cvr sequence was repeated times with the profile aaaabnaaba. in contrast, in strains from the five other territories, the cvr sequences contained only repeats with the signature aabnaaba (table ). both cvr signatures were different from the strains tan /moshi, bur / , bur / and ug / [ ] [ ] [ ] . no. strains positive for asfv using the pcr assays a pcr assays were performed on the samples positive for ppa pcr sequence analysis of the intergenic region between i r and i l genes and the ep r gene amplification of the ep r gene (encoding cd v protein) was performed, and pcr amplicons of strains from territories were successfully sequenced. comparative analysis of the sequences obtained was carried out together with other asfv sequences retrieved from the genbank database and previously characterized as serogroups. in this study, the phylogenetic analysis showed that the south kivu strains belonged to serogroup and were grouped with the uganda strain (km ), the only available serogroup in the gen-bank. this research suggests that the strains from this study may have a similar hemadsorption inhibition (hai) characteristics as the only known strain serogroup (fig. ) . the analysis of whole-genome sequences of asfv has facilitated identification of several regions containing tandem repeat sequences, important for discriminating between closely related asfv strains and for predicting the origin of the virus [ ] . in this study, the intergenic region between the i r and i l genes was analyzed for strains from the territories studied. the south kivu asfv strains were compared to the kenyan strain (ay ), which was identified from a domestic pig. the sequence alignment showed an indel of bp ( ′-cctatatacctataatcttataccctataa tct- ′) between nucleotide position to (fig. ). african swine fever constitutes the major obstacle to the development of the pig industry in the drc, with sporadic outbreaks occurring across various areas throughout the year [ ] . despite recurrent occurrence of suspected asf outbreaks in south kivu province, information on the virus characterization remains scarce. to determine the prevalence of asf and genotypes of asfv in circulation in the south kivu province, a study was carried out in the south kivu province from january to august , a period with no report of asf outbreaks a b fig. phylogenetic relationships of p and p genotypes. the evolutionary history was inferred by the maximum likelihood method based on the kimura -parameter model [ ] . phylogeny was inferred following bootstrap replications, and the node values show percentage bootstrap support. scale bar indicates nucleotide substitutions per site. scale bar indicates nucleotide substitutions per site. a p genotypes. the analysis included b l (p ) sequences from this study (plain circle ) and sequences from the genbank database. the genbank accession numbers for the different b l (p ) genes are indicated in parenthesis. b p genotypes. the analysis involved e l (p ) gene sequences of african swine fever viruses from this study (black diamond ◆) and sequences from the genbank database. the sequences for the different b l (p ) and e l (p ) genes are starting by genbank accession numbers or cases in the sampled area [ ] . we conducted a cross-sectional study in of the districts of the province and pig blood from smallholder pig farms were screened for presence of the asfv antibody and viral genome using indirect enzyme linked immunosorbent assay (elisa) and polymerase chain reaction (pcr), respectively, on asymptomatic domestic pigs. we found that % of pigs contained asfv antibodies whereas virus dna was present in . % of pigs. sequence analysis revealed that all the asfv detected from asymptomatic pigs belonged to the genotype ix. continuous characterization of asfv strains is key in endemic regions to better understand disease outbreak patterns and map the different strains according to their geographical regions, in which they circulate [ ] . the present study was targeting domestic pigs showing asf clinical signs with the aim to characterize asfv in symptomatic animals in the south kivu province. we confirmed the presence of asfv in domestic pigs with clinical signs of asf in the six studied territories of south kivu province: kabare, kalehe, fizi, mwenga, uvira and walungu. although this study was not designed to determine the prevalence of asfv, low rate of infection was observed in mwenga and kalehe ( and . %, respectively), whereas the highest infection rates were registered in walunga and uvira ( and . %, respectively). in our previous study which included asymptomatic pigs [ ] , walungu had the highest prevalence of asfv ( . %) while the lowest asfv prevalence was found in kalehe ( . %). the overall low infection rate may be attributed to the sensitivity of the assay used. indeed, the conventional pcr method used in this study is less sensitive than other molecular methods such as nested-pcr [ ] and real-time pcr [ ] and may fail to detect potentially positive samples containing very low amount of viral genetic material. in addition to the low sensitivity of the conventional pcr used, other conditions may have contributed to the observed low prevalence including: (i) most pigs sampled may not have been infected or may have low virus load not detectable by the pcr technique used; and (ii) suspected pigs may have been affected by other diseases and conditions with similar symptoms to asf such as porcine reproductive an respiratory syndrome, porcine dermatitis and nephropathy syndrome, salmonellosis. our results confirmed asf viral infections in pigs with clinical signs of asf in the south kivu province. from the ppa-pcr positive samples, , and samples were successfully amplified and sequenced for b l (p ), e l (p ) and b l (cvr) genes, respectively. the combination of these three viral regions is to ensure a high-level resolution for asfv discrimination. the p genotyping study corroborated the p analysis. both p and p phylogenetic analyses clustered asfv strains in circulation in symptomatic domestic pigs during the december -january outbreaks in the south kivu province with asfv genotype x, which is the major genotype associated with asf outbreaks throughout burundi, in some parts of tanzania, kenya and uganda [ , ] . genotype x has been reported to be a sylvatic cycle-associated genotype that include asfv identified from domestic pigs, warthogs as well as ticks in these three countries [ , , ] . furthermore, alignment of the bp long sequence from the variable ′-end of the b l (p ) gene in the south kivu viruses showed % identity with asfv strains from burundi (data not shown). it is a possibility that viruses in this study may originate from, or could have expanded to burundi. this scenario seems plausible as the south kivu province is bordering burundi through the river rizizi and lake tanganyika, and uncontrolled cross-border movements of pigs and pork products are observed in the region and may constitute a major route of transmission of asf in this endemic area [ ] . our current result contrasts with our previous finding of circulating asfv strains of genotype ix in asymptomatic domestic pigs in the studied area during a period with no asf outbreaks or cases [ ] . it is unlikely that data from both studies suggest that asfv of genotype ix may not cause disease in pigs whereas [ ] . although the report did not have any cases in the south kivu province, it identified asfv of genotype i in the neighboring province of maniema. nevertheless, further investigation in relation to both the host and virus genetics will be important to understand our findings. we are currently working on the lab-isolation of viruses of genotype ix and x in circulation in asymptomatic pigs and symptomatic pigs, respectively, for complete genome sequencing and comparative genomic analysis. data obtained will improve our understanding of this contrasting finding in pigs within the south kivu province. to the best of our knowledge, this is the first report of asf virus genotype x in the dr congo. as all the strains were p /p genotype x, we further characterized them at a higher resolution using the intra-genotypic central variable region (cvr) of the bl l gene. based on the tetrameric repeat sequences (trs), our analysis identified two different cvr variants, clustering the strains into two subgroups. subgroup was composed only of strains from uvira characterized by trs whereas all other strains formed the subgroup and had only trs. the profile of the subgroup (aabnaaba) was almost identical to the cvr amino acid sequence of uganda / (aabnbaba), having the b code (cadt) in place of a at the th repeat [ ] . the number of trs repeat observed is relatively small compared to reports from some studies in the same geographic region describing the trs motif repeated over to times [ , ] . however, mulumba-mfumu et al. also observed this sequence repeated only or times in some dr congo strains [ ] . the two cvr variants found in our study were different from the previously reported variants in dr congo asfv strains [ ] and to any other known viruses causing outbreaks or asf cases, thus suggesting that the asfv genotype in circulation in the south kivu province of dr congo identified in this study may be unique [ , ] . within the vaccine field, it has been suggested that protective immunity is serotype-specific, as defined by asfv hemadsorption inhibition (hai) serological assay, with viruses within a serogroup cross-protecting against one another [ , ] . the hai assay can be used to type asfv of a given genotype into distinct and individual serogroups, based on the asfv proteins cd v (ep r) and/or c-type lectin (ep r). thus, hai-based serogroup classification has been suggested as a better correlate for in vivo cross-protection among strains compared to the p genotyping [ ] . in our study, we obtained cd v sequences of strains from territories and comparative sequence analysis revealed that they were all identical. moreover, phylogenetic analysis showed that the uganda strain (genbank acc. no. km . ), which represents the only member of the serogroup , was closest related to the south kivu viruses, suggesting that the asfv strains, identified in this study, may belong to serogroup . the high bootstrap value of % grouping the south kivu strains with the uganda serogroup and the fact that strains from this study showed . % amino acid identity with the uganda serotype strain strongly support the genetic relatedness between these two groups. it is worth noting that strains of serogroups and have been reported in drc [ ] . overall, our data showed that these south kivu asfv strains are serologically different from other strains reported so far. analysis of the intergenic region between the i r and i l genes has previously been used for distinguishing between closely related asfvs [ ] . characterization of fig. partial nucleotide sequence alignments of the intergenic region between i r and i l genes. sequences of african swine fever virus (asfv) strains from the south kivu province, eastern drc, showing tetrameric repeats of representative genotypes, including a reference sequence of a virus isolated in in kenya (kenya ; genbank accession no. ay . ). the indel that results from the insertion of the nucleotide sequence cctatatacctataatcttataccctataatct in the asfv from kenya is boxed this intergenic region genes did not identify any genetic diversity among the south kivu strains. however, all the strains analyzed had high sequence identity with the kenyan strain (genbank acc. no. ay ) but lacked an insertion of bp. indels have also been reported in a similar analysis [ ] . altogether, our study provided evidence of circulating asfv genotype x which were antigenically related to serogroup in domestic pigs with clinical signs of asf in eastern drc. the genotyping approach was also supported with the hai serotyping for improved diversity analysis and finer discrimination of asfv strains. this represents the first report of asfv genotype x in drc. in this study, asfv isolated from symptomatic domestic pigs in the south kivu province of the democratic republic of congo were characterized for the genetic diversity. all the asfv strains analyzed in this study belonged to the p genotype x and the cd v serotype . this is the first report of 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viruses from outbreaks in peri-urban kampala, uganda development of a nested pcr and its internal control for the detection of african swine fever virus (asfv) in ornithodoros erraticus molecular diagnosis of african swine fever by a new real-time pcr using universal probe library african swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site transmission routes of african swine fever virus to domestic pigs: current knowledge and future research directions prevalence of african swine fever virus in apparently healthy domestic pigs in uganda molecular characterization of african swine fever virus in apparently healthy domestic pigs in uganda publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we acknowledge the biosciences eastern and central africa authors' contributions bnp collected samples for dna isolation, performed laboratory work and wrote the manuscript; jro supervised the study, conceived and designed the study, and edited the manuscript; ckt performed the laboratory work and edited the manuscript; emm performed the laboratory work and sequence analysis; ebb conceived and designed the study and edited the manuscript; ls supervised the study and edited the manuscript; rp supervised the study, performed sequence analysis, wrote and edited the manuscript. all authors read and approved the manuscript. this work was funded by the biosciences eastern and central africa -international livestock research institute (beca-ilri) hub, through africa biosciences challenge fund (abcf). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate see material and methods section. not applicable. the authors declare that they have no competing interests.author details department of molecular biology and biotechnology, pan african university, institute of basic sciences, technology and innovation, nairobi, kenya. key: cord- -qylm koe authors: müller, marcel a; van der hoek, lia; voss, daniel; bader, oliver; lehmann, dörte; schulz, axel r; kallies, stephan; suliman, tasnim; fielding, burtram c; drosten, christian; niedrig, matthias title: human coronavirus nl open reading frame encodes a virion-incorporated n-glycosylated membrane protein date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qylm koe background: human pathogenic coronavirus nl (hcov-nl ) is a group (alpha) coronavirus commonly associated with respiratory tract infections. in addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. our study focuses on hcov-nl open reading frame (orf ) which is a highly conserved accessory protein among coronaviruses. results: in-silico analysis of the amino acid sequence of hcov-nl orf predicted a triple membrane-spanning protein. expression in infected caco- and llc-mk cells was confirmed by immunofluorescence and western blot analysis. the protein was detected within the endoplasmatic reticulum/golgi intermediate compartment (ergic) where coronavirus assembly and budding takes place. subcellular localization studies using recombinant orf protein transfected in huh- cells revealed occurrence in ergic, golgi- and lysosomal compartments. by fluorescence microscopy of differently tagged envelope (e), membrane (m) and nucleocapsid (n) proteins it was shown that orf protein colocalizes extensively with e and m within the ergic. using n-terminally flag-tagged orf protein and an antiserum specific to the c-terminus we verified the proposed topology of an extracellular n-terminus and a cytosolic c-terminus. by in-vitro translation analysis and subsequent endoglycosidase h digestion we showed that orf protein is n-glycosylated at the n-terminus. analysis of purified viral particles revealed that orf protein is incorporated into virions and is therefore an additional structural protein. conclusions: this study is the first extensive expression analysis of a group hcov-orf protein. we give evidence that orf protein is a structural n-glycosylated and virion-incorporated protein. the human coronavirus (hcov)-nl constitutes one of four circulating prototypic human coronaviruses (cov) [ ] . hcov-nl infection causes upper and lower respiratory tract disease and is globally widespread, particularly among children under the age of six years [ ] [ ] [ ] . it was shown to be associated with croup [ , ] . cov belong to the nidovirales. the cov genome consists of a to kb positive single-stranded rna which is '-capped and '-polyadenylated [ ] . the genome of hcov-nl comprises , nt and has a gene organization conserved in all cov, i.e., gene a/b, spike (s), open reading frame (orf ), envelope (e), membrane (m) and the nucleocapsid (n) gene. cov virions consist of a nucleocapsid core surrounded by an envelope containing three membrane proteins, s, e, and m. cov assemble and bud at membranes of the endoplasmic reticulum (er)-golgi intermediate compartment (ergic) [ , ] . while the budding site of several cov has been localized at the ergic, the viral surface proteins can also be found in downstream compartments of the secretory pathway [ ] . m localizes predominantly in the golgi apparatus [ , ] , s is found along the secretory pathway and at the plasma membrane [ , ] , and e is detected in perinuclear regions, the er and golgi [ ] [ ] [ ] . s and m are typically glycosylated and it was shown that glycosylation plays an important role in the generation of bioactive protein conformations and influences fusion activity, receptor binding, and antigenic properties of cov [ ] [ ] [ ] [ ] . in addition to the s, e, m and n protein genes, the structural gene portion of cov genomes contains a variable number of accessory orfs. because these accessory orfs are not shared between different cov groups, they are also referred to as group-specific orfs [ ] . proteins encoded by group-specific orfs of different cov have been shown to influence pathogenesis, virus replication, or host immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] . others may be dispensable for virus replication in cultured cells of primate or rodent origin, as well as in rodent models [ , , ] . the orf is the only accessory orf conserved in all covs [ ] . most investigations of its functionality have been done on the example of sars-cov orf a. the sars-cov orf a protein is expressed in infected cells and patient sera contained antibodies reactive with recombinant orf a antigen. the n-terminal ectodomain was able to induce virus-neutralizing antibodies in rabbits [ ] . sars-cov orf a protein is a triple-spanning membrane protein with a similar topology as the m protein, and is integrated into virions [ ] . moreover, truncated forms were discovered for recombinantly and virally expressed orf a protein which could also be detected in virions [ ] . unlike the m protein it is not n-glycosylated but o-glycosylated and it was shown to interact with e, m and s protein [ , [ ] [ ] [ ] . subcellular localization of orf a protein was found to be at the golgi complex and the plasma membrane where it was also internalized by endocytosis [ ] . orf a protein was shown to induce apoptosis [ ] and cell cycle arrest [ ] and to up-regulate expression of fibrinogen in lung epithelial cells [ ] . although small interfering rnas targeting the orf a-specific viral subgenomic rna were able to reduce viral replication [ ] , deletion of orf a from an infectious cdna clone had no effect on viral replication in cell culture and mice [ ] . moreover it has been demonstrated that sars-orf a protein forms a homotetramer through inter-protein disulfide bonds, functionally working as a potassium ion channel that modulates virus release [ ] . very recently it was shown that the orf a protein disrupts the architecture of the golgi apparatus and might thus be responsible for the formation of vesicular structures in which virus replication takes place [ ] . sars-cov as a member of cov group b (beta) is only distantly related to the human cov-nl , a member of group b (alpha). for the orf protein of group (alpha) covs investigations have focused on the porcine epidemic diarrhea virus (pedv, group b, alpha) and transmissible gastroenteritis virus (tgev, group a, alpha) that cause enteropathogenic diarrhea in swine [ ] . it was shown that virulence of these viruses could be reduced by altering the orf gene through cell culture adaptation [ , ] . for hcov-nl , preliminary experiments suggested that deletion of orf had little influence on viral replication in cell culture [ ] . however, the closely related hcov- e has a homologous gene named orf that is split into two orfs ( a and b) in cell culture but maintained in all circulating viruses. this suggests an in-vivo function that may not be necessary for viral replication in cell culture [ ] . in the present study we characterized the orf protein of hcov-nl . we analyzed the expression and subcellular localization of the orf protein in virusinfected cells and cells transfected transiently with orf protein-expressing plasmids. we determined the topology of the orf protein, characterized its glycosylation, and showed that the orf protein is a structural protein incorporated into viral particles. the hcov-nl genome contains an open reading frame (orf ) situated between the s and e genes (figure a) . nucleic acid sequence alignments with homologous genes of other cov from groups alpha, beta and gamma yield nucleotide identities between , % and , % (table within figure a ). amino acid alignments showed highest levels of similarity ( %) and identity ( %) between hcov-nl orf protein and the homologous protein of hcov- e [ ] . a constant level of similarity was observed across the whole protein. in-silico analysis of potential glycosylation sites and membrane topology suggest properties similar to sars-cov orf a protein ( figure b and table ). hcov-nl encodes a aa protein (approximately kda) with three putative transmembrane domains at aa positions - , - and - , respectively (tmhmm analysis). it has three potential n-glycosylation sites (nxs/t) at aa positions , and , of which probably only the first is used because the sites at positions and are located inside the predicted transmembrane domains. no o-glycosylation sites are predicted. nearly half of the protein ( of aa) forms a hydrophilic c-terminus. these findings are in concordance with earlier data comparing sars-cov a-like cov proteins [ ] . to analyze the expression of orf protein during viral replication, colon carcinoma cells (caco- ) and rhesus monkey kidney cells (llc-mk ) cells were infected with hcov-nl and an immunofluorescence assay (ifa) was done after two and four days, respectively. a rabbit polyclonal antiserum raised against a peptide representing the c-terminal aa - of the predicted orf protein yielded fluorescence in the cytoplasm as shown in figure a and b (upper panel). because colocalization of sars-cov orf a protein with the ergic has been reported [ , ] , the same cells were counterstained with a murine monoclonal antibody against the ergic marker protein. [ ] , it was analyzed whether hcov-nl orf and m proteins were located in the same compartment. as shown in figure b (bottom panel), a strong colocalization was also seen for anti-nl m and anti-ergic signals. after showing that the orf protein can be found within the ergic compartment in infected cells we were interested in which other cellular compartments an isolated overexpressed orf protein can be detected. therefore we transfected huh- cells and stained the orf protein with the specific antiserum and co-stained different cellular compartments with specific antibodies (mouse-anti-ergic , mouse-anti-golgi k, goat-anti-lamp- for trans-golgi/lysosomes). as shown in figure the recombinant orf protein can be detected in all major compartments of the secretory pathway ( figure a for ergic, b for golgi and c for trans-golgi and lysosomes). these localizations are in concordance with recently published data on the homologous sars-cov orf a protein that is responsible for golgi membrane rearrangement [ ] . for sars-cov orf a protein, colocalization with the structural proteins s, e, and m, but only partial colocalization with n has been suggested [ ] . to investigate colocalization of nl -orf protein with structural proteins, an expression plasmid containing orf with an n-terminal flag-tag epitope was co-transfected with vectors coding for green fluorescent protein (gfp) fused to hcov-nl e, m and n proteins, respectively. expression of proteins with correct molecular weights was confirmed by western blot analysis (data not shown). the ergic compartment was stained in transfected cells as described above. as shown in figure , gfp-e and gfp-m both showed extensive colocalization with flag-orf protein. protein complexes were localized predominantly within the ergic, represented by white areas in figure . gfp-n had primarily a cytosolic distribution but there were small areas of colocalization with flag-orf protein, within the ergic compartment. all experiments were done in huh- cells supportive of hcov-nl replication, but these same findings were also confirmed in another cell line, human embryonic kidney (hek)- t (data not shown). to rule out altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated using flag-orf protein in combination with ha tagged e, m and n proteins in hek- t cells ( figure b ). again, colocalization of orf protein with e and m protein and, to a far lesser extent, with n protein was seen. posttranslational modification of the orf protein in hcov-nl -infected llc-mk cells was analyzed by western blot. the m protein which had a very similar predicted molecular mass of kda (table ) served as a control. as expected, the m protein and a protein corresponding to orf protein migrated at corresponding heights in western blots ( figure a ). both proteins showed additional bands at slightly higher molecular mass, consistent with posttranslational modification. in contrast to virus-infected cells, cells overexpressing orf protein from plasmid with an n-terminal flag epitope showed only a single band in western blot whose migration was consistent with the hypothetical unglycosylated form ( figure b, left panel) . it was assumed that glycosylation at the predicted n-glycosylation site at position (table ) might be ablated in the overexpressed protein, due to presence of the n-terminal epitope tag. indeed, recombinant orf (rorf ) protein without any tag and overexpressed in the same cells from the same vector showed both forms, identical to those observed in virus-infected cells ( figure b , right panel). to determine whether n-terminal glycosylation was to be expected at position , the membrane topology of the n-terminus was examined next. based on our in-silico analyses and in agreement with reports on sars-cov orf a protein [ ] , we hypothesized that the hcov-nl orf protein n-terminus reached the er lumen and was eventually exposed on the cell surface. for confirmation, n-terminally flagtagged orf protein was overexpressed in hek- t cells and stained by ifa using monoclonal antibodies against the flag tag, or alternatively, a polyclonal antibody against a peptide representing the orf protein c-terminus. as shown in figure , a perinuclear distribution of fluorescence was observed with both antibodies in permeabilized cells. in non-permeabilized cells, only the anti-flag antibody yielded fluorescence at cell surfaces. unfortunately, there was no complete overlap of signals from both antibodies in fully permeabilized cells in merged fluorescence pictures, most likely due to additional non-specific recognition of non-viral epitopes by the polyclonal antibody against the orf protein cterminus. for this reason a clear intracellular localization of the c-terminus in relation to the er/golgi membrane could not be formally determined. however, it could be concluded that the n-terminus of the orf protein was facing towards the extracellular space. according to in-silico predictions the orf protein contained three putative n-glycosylation sites at positions , and ( figure b, table ). only position was considered a possible n-glycosylation target, as the other two positions would be located within the membrane. in a vector expressing orf protein with a c-terminal v tag, asparagine (n) at position was changed into glutamine (q). in-vitro translated s-radiolabelled proteins with and without the exchange were treated or not treated with endoglycosidase h prior to sds-page analysis. sars-cov m protein served as the control because it had been shown previously to be nglycosylated exclusively at position four [ ] . in-vitro translated nl protein orf with and without the v tag, but not the same protein with an n q exchange, showed a second band of increased molecular weight in sds-page that disappeared upon endoglycosidase h treatment (figure ). in the same way as for sars-cov m-protein, deglycosylation did not change the apparent molecular weight of the lower band, verifying absence of any further active glycosylation sites. our data suggested that the orf protein was a glycosylated protein that colocalized with structural proteins in the ergic. protein orf might thus constitute a structural protein itself. to assess if the orf protein was incorporated into virions, viral particles were purified by sucrose gradient ultracentrifugation. after centrifugation, the gradient was divided into ten fractions and infectivity within each fraction was determined by plaque assay (figure ). only fractions to correlating with a sucrose density of % to % contained infectious particles with a peak of . × e pfu/ml in fraction (sucrose density - %). subsequent western blot analysis identified the same pattern of accumulation within the gradient for the orf protein as for the structural m and n proteins. anti-actin staining excluded cellular contamination in these fractions. it was concluded that hcov-nl orf protein was incorporated into viral particles. the orf protein and its homologues are conserved among covs [ ] . although identities on nt and aa level are low, most are predicted to be triple membrane-spanning proteins [ ] . while it has been suggested that orf homologues are dispensable for replication in cell culture, mutations of orf homologues in transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) lead to attenuation of virus in-vivo in pig models [ , , ] . because the sars-orf a protein underwent positive selective pressure during the human epidemic in / [ ] , an important function in-vivo can be assumed for the sars-cov orf a protein as well. unfortunately, it remains difficult to characterize invivo functions of hcov-nl orf protein due to lack of any animal model. however, it is interesting to note that across all strains of hcov-nl characterized so far, there are no mutations in the orf amino acid hcov-nl orf protein with and without a c-terminal v tag, and with an n q exchange in the tagged version was in-vitro translated in presence of s-methionine. sars-cov m protein without a tag was translated in the same system as a control. proteins were digested with endoglycosidase (endo h) as indicated below each lane, subjected to sds-page, and visualized. note the removal of the bands of increased molecular weight for the control and orf proteins, but not for the orf protein with an amino acid exchange at the hypothetical n-glycosylation site. note also that extent of size reduction for the sars-cov m protein, which is known to have one n-terminal n-glycosylation site, is the same for the nl orf protein. figure identification of nl -orf protein as a structural viral protein by sucrose gradient ultracentrifugation. viral supernatant was purified via subsequently centrifugation on two discontinuous and one continuous sucrose gradients of % to % (w/v) sucrose. the continuous cushion was divided into ten fractions as indicated in part (a). after centrifugation of each fraction through % sucrose cushions, the resulting pellets were analyzed for infectious particles by plaque assays. resulting virus titers are indicated on the y-axis in part (a). (b), fractions - were subjected to western blot analysis using specific rabbit antibodies against orf , m and n protein ( : ; : , and : , , respectively). to exclude cellular contaminations in the fractions a western blot using mouse-anti-actin ( : , ) was performed. note the colocalization of the orf protein in the same gradients as the known structural proteins m and n. sequence [ , ] . conservation of orf matches results by donaldson et al., showing that virus production in human airway epithelium was reduced when the orf protein was replaced by gfp [ , ] . it has thus been suggested that protein orf might serve functions involved in viral egress which are relevant for spreading in airway epithelium but not in simpler cell culture [ ] . results from this study, in particular the subcellular localization of orf protein along the secretory pathway (ergic, golgi, plasma membrane), the colocalization of nl -orf protein with other structural proteins in the ergic and the inclusion of the orf protein in virions give support for a hypothetical function within the viral assembly and budding process. a range of further hypotheses can be derived from earlier investigations into protein orf functions. these include antigen decoy functions as suggested for sars-cov orf a [ ] , interference with the regulation of expression of nfb-dependent cytokines [ , ] and fibrinogen [ ] , and finally the modulation of s protein mediated endocytosis [ ] or an hypothesized down-regulation of the expression of s protein on the cell surface [ ] . rhesus monkey kidney llc-mk cells (atcc: ccl- ), human embryonic kidney hek- t cells (attc: crl- ), human hepatocellular carcinoma cell line (huh- , jcrb kindly provided by antoine a. f. de vries, lumc, leiden) and colon carcinoma caco- cells (atcc: htb- ) were grown at °c and % co in dulbecco's modified eagles medium (dmem; gibco, karlsruhe, germany) containing % fetal calf serum, mm l-glutamine and u of penicillin/ml and u streptomycin/ml (paa laboratories, linz, austria). all cells were tested negative for mycoplasms by pcr as described elsewhere [ ] . if not stated otherwise materials were provided from roth, karlsruhe, germany. for virus stock production either caco- or llc-mk cells were inoculated with hcov-nl ( th passage amsterdam strain i; accession no. nc_ ) at a multiplicity of infection (moi) of . and infected cells were cultured at °c and % co for five to seven days before harvesting. after centrifugation at , × g for min supernatant was aliquoted and stored at - °c. titers were determined by plaque assay performed as described elsewhere [ ] . briefly, after incubation of the plaque assays at °c and % co for four days cells were fixed with % formaldehyde, stained with crystal violet solution and results were interpreted as described previously [ ] . for first strand cdna synthesis total rna was extracted from infected cells five to seven days post infection (dpi). reverse transcription was performed as described elsewhere [ ] using oligo(dt) primers (fermentas, st. leon-roth, germany). in order to recombinantly express hcov-nl proteins orf , e, m and n we cloned the different genes into a variety of expression vectors. for generation of gfp-constructs pcr was performed with the following specific primers listed in table table ). in this case pcr products were digested with restriction endonucleases ecori and noti (fermentas) before cloning into the pcaggs vector (also digested and additionally dephosphorylated before use). generally, pcr was performed with platinum® taq dna polymerase high fidelity (invitrogen, karlsruhe, germany), and conditions were as follows: °c for min, followed by cycles of °c for s, primer specific temperature for s, and °c for s, with a final extension at °c for min. the different genes were cloned into pcdna . /v -his-topo (eukaryotic expression and invitro translation) and pcdna . /nt-gfp-topo (eukaryotic expression) with the help of topo expression kits (invitrogen) according to the manufacturer's instructions. cloning of flag-tagged orf into the pcaggs vector was done conventionally with t ligase (invitrogen) according to suppliers' description. correct cloning was confirmed by sequencing (abi prism , ; applied biosystems, foster city, usa). the generation of a polyclonal antiserum against orf was done with the help of keyhole limpet hemocyanin (klh) coupled peptides. two peptides were synthesized corresponding to aa positions - and - (eurogentec, seraing, belgium). immunization was per- formed in-house. briefly, a chinchilla rabbit was immunized four times with μg of a mixture of the two klh coupled peptides and sera were tested as suggested by the manufacturer by enzyme-linked immunosorbent assay (elisa) using the corresponding uncoupled peptides. we then tested serum with ifa using infected llc-mk cells (figure ) as well as with prokaryotic recombinant proteins with the help of dot blot and western blot analysis (data not shown). the bleeding for the applied anti-orf serum was carried out days after the fourth injection and sera were used directly. typically, × caco- or llc-mk cells were seeded on glass slides in a -well plate and infected with hcov-nl as described above. two to four days after infection the cells were fixed with paraformaldehyde ( %) for min and permeabilized with . % tritonx (merck, darmstadt, germany) for min. afterwards the cells were washed with pbs again and then incubated with the primary antibody, diluted : in sample buffer (euro-immun, lübeck, germany), at °c for h. the ergic was stained with the help of mouse-anti-ergic (axxora, grünberg, germany). in order to stain the golgi apparatus we used a mouse-anti-golgi k (sigma-aldrich, munich, germany). for staining of the trans-golgi network and lysosomal compartment we applied a goat-anti-lamp- antibody (santa cruz biotechnology, heidelberg, germany). secondary detection was done with fluorescein isothiocyanate (fitc) or cyanine (cy )-conjugated goat-anti-rabbit as well as with rhodamine or cy -conjugated goat-anti-mouse or donkey-antigoat antibody (dianova, hamburg, germany) at °c in a wet chamber for min. slides were mounted and analyzed by clsm meta laser confocal microscope (zeiss, jena, germany). western blot analysis of viral proteins was done as described elsewhere [ ] . for titration of the different rabbit antisera we used hcov-nl cell lysate generated from llc-mk infected cells five to seven dpi (~ × cells/blot) for western blotting and incubated the produced nitrocellulose strips with the different rabbit antisera (pre-immune sera as negative control) at dilutions ranging from : up to : , (data not shown). generally, cells were lysed in ripa lysis buffer ( mm nacl, % igepal ca- , . % sodium deoxycholat, . % sds, mm tris (ph . )) and separated on a % sds-page gel. western blotting was performed by using anti-orf , anti-m, anti-n at dilutions : , , : , and : , respectively. secondary detection was done with the help of supersignal® west dura extended or femto chemiluminescence substrate (pierce biotechnology, rockford, usa). transfections of hek- t and huh- cells with eukaryotic expression vectors containing the fusion genes gfp-e, gfp-m, gfp-n, ha-e, ha-m, ha-n and flag-orf were performed with the help of fugene hd (roche, basel, switzerland) transfection reagent as described above using -well plates provided with glass slides. after a h incubation at °c and % co transfected cells were washed with pbs and fixed with paraformaldehyde ( %), permeabilized with tri-tonx and incubated with rabbit-anti-flag (sigma) and mouse-anti-ergic (axxora) primary antibodies, both diluted : with sample buffer (euroimmun). secondary detection was performed with cy -conjugated goat-anti-rabbit ( : ) and cy labelled goatanti-mouse ( : ) antibodies (dianova). slides were mounted and analyzed by confocal laser scanning microscopy. for western blot analysis of recombinant orf proteins (flag-orf , rorf ) transfections were performed in -well plates using fugene hd transfection reagent. transfection was performed with μg dna and μl fugene hd in μl dmem. transfected cells were washed three times with ice cold pbs and harvested for western blot analysis after incubation for to h at °c and % co . cell lysis was performed with ripa lysis buffer (~ × cells/ ml) containing protease inhibitor cocktail iii (calbiochem, san diego, usa) and benzonase ( u/ml) (novagen, madison, usa). after min incubation on ice samples were sonicated twice for s (branson sonifier , branson, danbury, usa) and centrifuged at , × g for min at °c. for detection of the different proteins we used rabbit-anti-flag (sigma, diluted : , ) or anti-orf antiserum ( : ) and incubated blots for to h at room temperature. as secondary antibody we applied a goat-anti-mouse or rabbit horseradish peroxidase (hrp)-conjugated antibody (pierce biotechnology) for h at room temperature. detection was performed by using supersignal® west femto chemiluminescence substrate (pierce biotechnology). plasmids pcdna . -orf -v /his, pcdna . -orf -n q-v /his and pcdna . -orf were employed in the tnt t quick coupled reticulocyte lysate system (promega, mannheim, germany) according to the manufacturer's description. the proteins were metabolically labelled with [ s]methionine (ge healthcare, munich, germany) and translated in the presence of canine pancreatic microsomal membranes (promega). membranebound proteins were pelleted at , × g for min and resuspended in pbs. samples were split in half and incubated for h at °c with endoglycosidase h (endo h; new england biolabs, frankfurt, germany) or, as control, without additives. afterwards samples were subjected to sds-page. radioactive signals were visualized by exposing dried gels to bioimage plates, which were scanned by using a bioimager analyzer (bas- , ; fuji). purification of viral particles was performed by sucrose gradient ultracentrifugation as described elsewhere [ ] . briefly, ml viral supernatant from infected caco- cells was cleared from cell debris dpi and subsequently applied onto two discontinuous and one continuous sucrose cushion of % to %. the continuous cushion was divided into ten fractions and viral particles were pelleted by ultracentrifugation through a % sucrose cushion. virus pellets were resuspended in μl pbs and stored at - °c. prediction of protein topology and subcellular localization was done by netnglyc, netoglyc, tmhmm http://www.cbs.dtu.dk/services/, tmpred http://www.ch. embnet.org/software/tmpred_form.html, and prodiv/ topcons http://topcons.cbr.su.se/index.php. the alignments and a sequence identity matrix were done by using blast and mega (blosum; parameters p-distance and pair wise deletion). identification of a new human coronavirus human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry human coronavirus-nl infections in korean children human coronavirus nl , france. emerg 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severe acute respiratory syndrome coronavirus a protein is a viral structural protein the severe acute respiratory syndrome coronavirus a is a novel structural protein characterization of severe acute respiratory syndrome coronavirus membrane protein glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins a and m a novel severe acute respiratory syndrome coronavirus protein, u , is transported to the cell surface and undergoes endocytosis the a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e cells g phase cell cycle arrest induced by sars-cov a protein via the cyclin d /prb pathway the severe acute respiratory syndrome coronavirus a protein up-regulates expression of fibrinogen in lung epithelial cells inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, a/ b, a/ b and s severe acute respiratory syndrome-associated coronavirus a protein forms an 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transmissible gastroenteritis virus from britain with a deleted orf a differential stepwise evolution of sars coronavirus functional proteins in different host species hoek van der l: mosaic structure of human coronavirus nl , one thousand years of evolution severe acute respiratory syndrome coronavirus a protein is released in membranous structures from a protein-expressing cells and infected cells sars coronavirus accessory proteins augmentation of chemokine production by severe acute respiratory syndrome coronavirus a/x and a/x proteins through nf-kappab activation understanding the accessory viral proteins unique to the severe acute respiratory syndrome (sars) coronavirus logt van der jt: detection of mycoplasma pulmonis in experimentally infected laboratory rats by s rrna amplification plaque assay for human coronavirus nl using human colon carcinoma cells assessment of igg antibodies against yellow fever virus after vaccination with d by different assays: neutralization test, haemagglutination inhibition test, immunofluorescence assay and elisa susceptibility of different eukaryotic cell lines to sarscoronavirus coronavirus antibodies in african bat species. emerg infect dis human coronavirus nl open reading frame encodes a virion-incorporated n-glycosylated membrane protein this study was supported by the german ministry of education and research (project code "Ökologie und pathogenese von sars"), and the european commission (fp framework program no emperie). we are grateful to a. teichmann for excellent technical assistance. the authors declare that they have no competing interests. key: cord- -ghcwfcx authors: razanajatovo, norosoa h; nomenjanahary, lalaina a; wilkinson, david a; razafimanahaka, julie h; goodman, steven m; jenkins, richard k; jones, julia pg; heraud, jean-michel title: detection of new genetic variants of betacoronaviruses in endemic frugivorous bats of madagascar date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: ghcwfcx background: bats are amongst the natural reservoirs of many coronaviruses (covs) of which some can lead to severe infection in human. african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks. a recent study in south africa isolated a genetic variant closely related to mers-cov from an insectivorous bat. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic, no data exists concerning the circulation of covs in the island’s chiropteran fauna. certain malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. the purpose of our study is to detect and identify covs from frugivorous bats in madagascar to evaluate the risk of human infection from infected bats. methods: frugivorous bats belonging to three species were captured in four different regions of madagascar. we analyzed fecal and throat swabs to detect the presence of virus through amplification of the rna-dependent rna polymerase (rdrp) gene, which is highly conserved in all known coronaviruses. phylogenetic analyses were performed from positive specimens. results: from frugivorous bats, we detected coronaviruses from two endemic bats species, of which viruses were identified from pteropus rufus and one from eidolon dupreanum, giving an overall prevalence of . %. phylogenetic analysis revealed that the malagasy strains belong to the genus betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. conclusions: our findings suggest that covs circulate in frugivorous bats of madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. possible dispersal mechanisms as to how coronaviruses arrived on madagascar are discussed. coronaviruses (covs) are enveloped viruses with singlestranded positive-sense rna belonging to the subfamily coronavirinae in the family coronaviridae (order nidovirales). genomes of covs range from to kb and show high genetic diversity [ ] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [ ] . in mammals and birds, covs are associated with upper and lower respiratory illnesses or gastroenteritis. in humans, covs infections are commonly caused by hcov- e and hcov-oc which generally cause mild respiratory illnesses [ ] . a new cov that causes severe acute respiratory syndrome (sars-cov) emerged in humans in - and infected more than , individuals with mortality rates estimated at around % [ ] . the emergence of sars-cov and its mortality rate have raised the risk of a new pandemic that could threaten public health. for this reason, the scientific community invested considerable interest in the identification and characterization of covs especially within mammal reservoirs. subsequently, two novel human covs were discovered: hcov-nl in [ ] and hcov-hku in [ ] . in june , a third novel coronavirus named hcov-emc/ (renamed mers-cov) was isolated from patients presenting with acute respiratory distress and pulmonary inflammation [ , ] . studies which aimed to identify potential reservoirs of emerging human covs have revealed that the betacoronavirus sars-cov was closely related to covs detected in bats, specifically members of the genus (rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in chinese markets as bushmeat for human consumption [ ] [ ] [ ] . bats have since become a particular focus and a number of alphacoronavirus and betacoronaviruses have been identified in many frugivorous and insectivorous bat species and in many countries worldwide in asia, the americas and europe (see review from drexler et al. ) [ ] . genomic characterization of the recently discovered mers-cov showed that this virus belongs to the genus betacoronavirus and seems to be closely related to bat coronaviruses hku and hku isolated from bats (tylonycteris and pipistrellus) [ ] . african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks [ ] [ ] [ ] . some authors have reported the detection of bat covs from mainland africa [ ] [ ] [ ] [ ] [ ] . a recent study in south africa detected a genetic variant closely related to mers-cov from neoromicia zuluensis, an insectivorous bat [ ] . the authors hypothesize that mers-cov may have a common ancestors with covs borne by bats from africa. though madagascar is home to bat species ( insectivorous and frugivorous) of which are endemic [ ] [ ] [ ] , no data exists concerning the circulation of covs in malagasy bats. certain bat species on the island can be frequently found in close contact with humans, particularly members of the family molossidae; these synanthropic species roost in human-occupied buildings, like houses, schools or hospitals [ ] , whilst frugivorous bats feed in the same fruit trees where people collect and consume fruits. moreover, hunting and consumption of bat bush meat, especially the larger frugivorous species of the family pteropodidae, is widespread on the island, bringing hunters, purveyors and consumers into contact with bats [ ] . in this study, we report the detection of covs amongst frugivorous bats in madagascar. our results demonstrated for the first time that covs belonging to the genus betacoronavirus are circulating amongst two endemic frugivorous bats species in madagascar. a total of bats belonging to endemic bat species of the family pteropodidae were captured and sampled: rousettus madagascariensis (n = ), pteropus rufus (n = ) and eidolon dupreanum (n = ) ( table ) . none of the throat swabs from any bat species (n = ) tested positive for cov, but . % ( / ) of fecal specimens tested positive for cov. prevalence within p. rufus, e. dupreanum and r. madagascariensis was respectively . % ( / ), . % ( / ) and % ( / ). all positive specimens originated from bats captured in the menabe region ( figure ). short amplicon sequences of bp in length of the rdrp gene were obtained for all pcr-positive animals, whereas larger fragment of bp sequences could only be obtained from seven of the pcr-positive animals. all amplicon sequences were aligned in-frame with a compilation of reference sequences from genbank for which collection-date data was available [ ] , giving final alignments containing different sequences of bp in length and different sequences of bp in length. gtr + i + g was identified as the optimal substitution model using jmodeltest v . . [ ] . multiple phylogenies were generated in beast using different combinations of model parameters, and the best models were selected using the tracer [ ] . bayes factor analysis employing marginal likelihoods, as detailed in [ ] . all parameter combinations produced identical, strongly supported tree topologies (data not shown). as has elsewhere been determined by lau et al. [ ] , bayesian skyline using a relaxed, exponentially distributed clock model was found to be the best fitting model for rdrp dated-tip phylogenies. the final phylogenetic analyses ( figure ) revealed that strains from madagascar are members of the betacoronavirus genus, rooting with hong kong strain btcov-hku (hm ) and kenyan strain ky (gu ), with posterior probabilities of . these lineages could be described as sars-like, and were uniquely affiliated with frugivorous bat hosts of the family pteripodidae. malagasy strains were sub-divided into three distinct clusters: two of which were closely related (clusters and ) and originating from p. rufus, and one more distantly related (cluster ) containing a strain detected from e. dupreanum and sequences previously detected from e. helvum in kenya [ ] . the malagasy covs were detected from bats captured in three different sites of the menabe region (west of madagascar). within cluster , strains were originated from bemanonga, mahabo and ankiliabo. within cluster , strains were originated from bemanonga and ankiliabo. the only virus detected belonging to cluster was detected in one bat captured in mahabo. overall, identities among malagasy covs ranged from to % at the nucleotide level and . to % at the amino acid level (data not shown). molecular dating estimates based on the bp fragment of the rdrp gene estimated the timescale of evolution of the coronavirus family to be thousands to tens of thousands of years, however dating estimates proved inaccurate, with broadly spanning hpd values at individual node positions. in the context of this study, we detected coronaviruses forming nine genetically distinct strains in two endemic malagasy frugivorous bat species. the overall prevalence ( . %) is consistent with those identified in studies elsewhere [ ] [ ] [ ] . thirteen viruses were detected from pteropus rufus and virus from eidolon dupreanum. we did not detect covs among the sampled r. madagascariensis. the detection of novel bat covs supports the observation that these viruses are diverse and have a nearly worldwide distribution [ , , , ] . we observed that all malagasy bat covs detected in the present study belong to a sars-related subgroup of the betacoronaviruses, with relatively close homology to btcov-hku [ ] . our strains displayed distinct clusters: clusters associated with p. rufus and cluster associated with e. dupreanum. it can be inferred from the results that multiple clusters of covs occurring within malagasy bat populations co-circulate and possibly in a syntopic manner. the high nucleotide and amino acid divergence between clusters and compared to the reference virus btcov-hku suggests previously undescribed genetic lineages. given the mobility of bats, and the especially long distances that can be travelled by colonies of fruit bats [ ] , these coronaviruses may be spread over a large region. however, host-genus-specific phylogenetic clustering (figure , inset) suggests likely host-specificity which may limit viral spill-over. thus, further molecular epidemiology studies would be required to fully understand the dispersal potential of covs amongst malagasy bats species. it is important to remember that, although all three of madagascar's fruit bat species were sampled, nothing is known of cov dynamics in tropical fruit bats, and many factors such as seasonality, bioclimate and the presence of other host species may have important influences on cov prevalence in these populations. more studies are needed in different locations including different species, particularly those with an insectivorous diet, to reveal a more comprehensive view of the diversity of these viruses in madagascar. since the strains of betacoronavirus identified from madagascar are closely related to those known from africa, some preliminary biogeographic considerations are in order. all three bat species analyzed herein are endemic to madagascar. the genus pteropus has a broad distribution from the australia-new guinea region west across the indian ocean to offshore islands of tanzania; it is unknown from the african continent. the genus eidolon is composed of two species: e. helvum occurring on the african mainland and offshore islands and e. dupreanum restricted to madagascar. based on a phylogeographical study, both species show broad panmictic population structure [ ] . further, these two taxa are estimated to have diverged from one another sometime in the late miocene or early pliocene [ ] . the genus rousettus is broadly distributed across africa and asia and the ancestral origin of the malagasy species, r. madagascariensis, is unresolved [ ] . as with the other two pteropodidae species occurring on madagascar, r. madagascariensis shows little genetic population structure and presumably broadly disperses across the island, which in turn has important epidemiological implications for these bats transmitting different zoonoses. although estimates of the most recent common ancestors (mrcas) proved inaccurate in our study, most likely as a result of a limited sequence availability from the identified viral strains, standard evolutionary analyses have estimated cov origins to date to somewhere between - , yrs. in the past [ ] [ ] [ ] . nevertheless, further investigations into the relevance of mrca prediction methodologies are required and a great level of caution must be employed in the interpretation of mrca data. . alternatively, viral lineages may have been imported to madagascar in recent history: while the vast majority of the island's bat fauna is endemic, a few species apparently disperse across the mozambique channel. probably the best example is the molossidae species mops midas, for which, based on genetic data, southern african animals are nested within malagasy populations [ ] . this bat makes its day roosts in rock crevices and may broadly occur synoptically at such sites with e. dupreanum and r. madagascariensis. these two fruit bat species are known to feed in the same fruiting trees with p. rufus [ ] , which would complete the cycle of how covs originated from africa mainland could be carried to madagascar and transmitted to different species of pteropodid bats. although we were not able to evaluate risk of human infection, the strains detected here may be considered as potential human pathogens, as bats are natural reservoirs of some pathogenic covs. isolation of malagasy covs using cell culture and molecular analysis of spike (s) gene could better evaluate risk of human infection. also, a longitudinal study amongst people who frequently handle live bats (e.g. bat hunters, bat bushmeat purveyors, and scientists), and who represent populations at higher risk of infection, would be interesting to establish possible cases of transmission to humans and public health risks. in our study, we confirm that covs are circulating in two species of endemic bats in madagascar. further work on cov diversity amongst the island's bat species, as well as aspects of the ecology and behavior of susceptible taxa, are needed to understand the origin, evolution and dispersal of these viruses across the island. to conclude, the results of our study demonstrate the need to develop research programs that aim at surveying viruses in the wild, especially in bats, in order to address possible emergence of zoonotic viruses within human populations. we sampled frugivorous bats in four different areas of madagascar: anjohibe, anosibe an' ala, menabe and toliara (table / figure ) based on known accessible colonies of roosting bats and sites where bats are frequently hunted and eaten by people. in the region of menabe, we selected different sites situated at a mean distance of km around mahabo to capture and collect specimens, while in the three other regions, sampling occurred at a single site. sampling was carried out under protocols approved and permitted by ministry of environment and forest (authorization # / , / , / , / and / ). fruit bats were captured by the use of mist-nets set near roosting sites (trees or caves) and with help of professional hunters [ ] . rectal and throat swabs were collected from each individual bat. all bats were identified according species specific morphological features well known by our field trained team (ecologist and veterinarian) and subsequently released. swabs were placed in viral transport media, almost immediately conserved in liquid nitrogen in the field and stored at − °c upon arrival at the laboratory in antananarivo. rna was extracted from specimens using the qiaamp viral rna minikit (qiagen, courtaboeuf, france) according to the manufacturer's protocol. briefly, total rna was extracted from μl of each sample and eluted in μl of qiagen ave elution buffer. the extracted rna was either immediately analyzed or stored at − °c until use. extracted rna was reverse transcribed to generate cdna by using the m-mlv reverse transriptase (invitrogen, california, usa) into a step reactions. first, μl of rna were mixed in a solution containing . μm of random hexamer primers (roche diagnostics, mannheim, germany), u/μl of rnase inhibitor ( units) and . μl of water, at °c for min, °c for min and °c for min. then, μl of rna issued from the first step was added to a mixture of . mm of each dntp (deoxynucleotide triphosphates), u/μl of reverse transcriptase m-mlv, x of buffer and . m of dtt (dithiothreitol), and incubated at °c for min and °c for min. pcr assay was performed to amplify the rna-dependent rna polymerase (rdrp) gene which is highly conserved in all known coronaviruses [ ] . the primers pair (forward ′-ggttgggactatcctaagtgtga- ′; reverse ′-c catcatcagatagaatcatcata- ′) was designed to amplify a bp fragment as described previously [ ] . reaction mixture was carried out using the gotaq/dntp mix, custom kit (promega corporation, madison, usa). briefly, μl of cdna was mixed with x of green gotaq flexi buffer, . mm of mgcl , . mm of each dntp, . μm of each primer, . u/μl of gotaq dna polymerase and . μl of water nuclease-free giving a final volume of μl. the thermocycling was performed under the following conditions: activation at °c for min and cycles of denaturation at °c for min, annealing at °c for min, extension at °c for min, and a final extension at °c for min. all negative samples were tested in a semi-nested pcr with the same pcr program and using the following pair of primers (forward ′-ggttg ggactatcctaagtgtga- ′; reverse ′-atcagata gaatcatcatagaga- ′). amplicons products were subsequently electrophorezed on a . % agarose gel and visualized using ethidium bromide under uv light. all specimens that showed a positive band at the expected size ( bp) were sequenced on both strands by beckman coulter genomics (essex, uk). from the sequences obtained from the bp, fragment, we designed new primers (reverse) that were strain specific for malagasy batcov ( ′-gatgacc tgtatattccca- ′ and ′-atgacctatacatacc catg- ). we then amplified a large fragment of the rdrp gene by using the consensus forward primer ′-gtgtacgctgctgatcctgctatgca- ′ [ ] . the following conditions were performed: °c for min and cycles of denaturation at °c for min, annealing °c for min, extension at °c for min, and a final extension at °c for min. the final size of sequences used for molecular dating was bp. sequences from the bp or bp fragments of the rdrp gene were cleaned and aligned with reference sequences collected from a literature search. alignment was performed using the translation alignment tool in geneious pro™ v. . . , created by biomatters (available from http:// www.geneious.com/), and the default clustalw cost matrix. the final alignment was respectively bp and bp in length for fragments bp and bp, and contained no free-end or internal gaps. from these alignments, the appropriate substitution model was identified in jmodeltest v. . . [ , ] . using the appropriate substitution model, * ^ iterations were run with or without the use of a base codon model, using different clock models, alternating between constant and bayesian skyline population size models, and seeding with uncorrelated log-normally distributed priors. trees were sampled every x ^ iterations, and analysis convergence was assessed in tracer v. . . [ ] (available from http://beast.bio.ed.ac.uk/tracer). all analyses converged after a % burn-in to give effective sample size values for all parameters superior to . bayes factor analyses were performed in tracer v . . , with bootstrap replicates to assess the relative performance of model selections on the generated phylogenies. after identification of an optimal model for phylogenetic classification and dating, two further independent analyses ( * ^ iterations, sampling every * ^ iterations) were run in beast, and all analyses were combined in logcombiner (beast package) with a burn-in of %, leaving only converged parameter estimates. the final phylogeny and mrca for fragments bp in length dates were extracted using treecombiner (beast package) and figtree v. . . 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methods for the study of bats identification of a novel coronavirus in bats genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was conducted in collaboration with the association madagasikara voakajy and the bangor university (darwin initiative project), a project examining aspects of emerging viruses in small wild mammals. the work in the toliara region was part of action concertée inter-pasteurienne (acip) research program. we would like to thank felicien herbert randrianandrianina of madagasikara voakajy and local hunters for their help in capturing bats. david a. wilkinson's post-doctoral fellowship was funded by "run-emerge", a european project funded by european commission under fp program. the authors declare that they have no competing interest.authors' contributions nhr and daw carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. lan coordinated the fieldwork, participated to the sampling collection, participated to molecular testing and drafted the manuscript. jhr participated in the design of the study and coordinated the fieldwork smg and rkj helped to draft the manuscript. jpgj participated in the design of the study and helped to draft the manuscript jmh: conceived, designed and coordinated the study and helped to draft the manuscript. all authors read and approved the final manuscript key: cord- -jh msz c authors: olagoke, olusola; quigley, bonnie l.; timms, peter title: koalas vaccinated against koala retrovirus respond by producing increased levels of interferon-gamma date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: jh msz c koala retrovirus (korv) is believed to be in an active state of endogenization into the koala genome. korv is present as both an endogenous and exogenous infection in all koalas in northern australia. korv has been linked to koala pathologies including neoplasia and increased susceptibility to chlamydia. a korv vaccine recently trialled in northern koalas improved antibody response and reduced viral load. this communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. the results showed that prior to vaccination, il- was expressed at the highest levels, with at least -fold greater expression compared to other cytokines, while cd mrna expression was significantly higher than cd mrna expression level. interferon-γ was up-regulated at both - and -weeks post-vaccination while il- was down-regulated at -weeks post-vaccination. korv is currently undergoing endogenization into the koala genome, thus offering an exciting opportunity to study retroviral endogenization in real time [ , ] . nine korv subtypes (korv-a to -i) have been identified with the subtypes a and b being the most studied [ ] . all northern koalas (queensland and new south wales) are thought to be harbour at least korv-a, a korv subtype which is both endogenous and infectious in these koala populations. korv infection has been linked to immune alterations, with infected southern koalas shown to have altered immune profiles [ ] and korv-b infected northern koalas shown to possess levels of immune dysregulation [ ] . korv has also been linked to increased susceptibility to diseases in koalas [ ] . we recently described antibody production and reduction in viral load following vaccination in koalas harbouring endogenous korv [ ] . briefly, koalas were vaccinated with a recombinant korv env protein along with a triadjuvant of poly i:c, polyphosphazine and host defence peptide . each animal received the vaccine subcutaneously on day zero with a booster dose delivered four weeks after. the vaccine was shown to lead to a significant increase in serum anti-korv igg levels and neutralising antibody titres. the antibodies were shown to be cross-reactive against exogenous korv subtypes. in the present study, we examined the expression of important koala cytokines, immune markers and host restrictions factors to determine their pre-and post-vaccination levels in northern koalas harbouring endogenous korv. to understand the expression profiles of key immune genes associated with vaccine response, we compared the pre-vaccination levels to -and -weeks post-vaccination levels. we chose to analyse the gene expression changes in a non-antigen stimulated model. genes targeted included nine cytokines (ccl l, interleukin open access *correspondence: oolagoke@usc.edu.au genecology research centre, university of the sunshine coast, sunshine coast, qld, australia (il)- β, il- , il- , il- , il- , il- a, il- and interferon gamma (ifn-γ), four host restriction factors (bst , isg , rsad and trim ) and two t-cell markers (cd and cd β). total rna was purified from µl whole koala blood using trizol ® ls (thermofisher), as per manufacturer's instructions. rna was treated with the turbo dna-free kit (thermofisher) before target gene transcripts were quantified using a custom nanostring probe panel (systems biology and data science, griffith university, australia). five koala housekeeping genes (actb, gapdh, hmg a, nckap l and stx ) were included in the panel for normalization and quality control. normalized transcripts were quantified pre-vaccination and converted to fold-change for post-vaccination analysis. expression levels of the target genes prior to vaccination were examined to gain an insight into the baseline immune genes expression of koalas harbouring endogenous korv. il- was by far the most abundant cytokine mrna expressed in these korv infected koalas (fig. a) , with over -fold higher expression compared to other cytokines. both il- and il- β were the next most abundantly expressed cytokines and they had comparable expression levels (fig. a ). the remaining cytokines tested (ccl l, il- , il- a, il- , il- and ifn-γ) had low, but detectable levels in all koalas investigated (fig. a) . additionally, all four host restriction factor genes (bst , isg , rsad and trim ) were expressed at quantifiable levels in all koalas (fig. b) . finally, cd gene expression levels were significantly lower than cd β gene expression levels (~ folds) (fig. c) . after vaccination, ifn-γ had the most pronounced expression change, with significant up-regulation at -weeks (log fold change = . ; p = . ) and continued up-regulation at -weeks (log fold change = . ; p = . ) post-vaccination (fig. a) . there were also a small but statistically significant down-regulation of il- a at -weeks post-vaccination (log fold change = − . ; p = . ) and il- by -weeks post-vaccination (log fold change = − . ; p = . ) (fig. a) . there were no significant changes in the mrna expression of the other cytokines tested (ccl l, il- β, il- , il- , il- , and il- ) at either -or -weeks post-vaccination, data not shown. in addition, none of the host restriction factors tested (bst , isg , rsad and trim ) had significant changes in their expression either at -or -weeks post-vaccination when compared to prevaccination levels. interestingly, the post-vaccination expression of cd mrna remained largely unchanged relative to pre-vaccination levels, while cd β mrna was slightly upregulated at -weeks post-vaccination (log fold change = . ; p = . ) (fig. b) . in this cohort of korv vaccinated and endogenously infected koalas, a small but significant increase in the expression of ifn-γ at both -and -weeks post-vaccination was observed, compared to pre-vaccination levels. while the biological impact of such sustained up-regulation is not fully known yet, a recent study showed that korv-positive southern (victorian) koalas had significantly lowered ifn-γ levels compared to korv-negative koalas [ ] . the authors of that study argued that such decreased ifn-γ levels may increase susceptibility to chlamydia and other opportunistic infections in korvpositive koalas. this work is the first to report an increase in ifn-γ in response to korv vaccination and, as such, suggests a means to counteract korv-associated reduction in ifn-γ levels in koalas. the antiviral properties of ifn-γ have been well documented. for instance, longlasting porcine circovirus vaccine efficacy was shown to be sustained, in part, by ifn-γ producing cells [ ] . ifn-γ has also been linked to b-cell differentiation into igg production [ ] . the increase in ifn-γ detected in this work may offer insight into the sustained anti-korv igg production and reduction in viral load reported in koalas following vaccination [ ] . a possible mechanism for the observed increase in ifn-γ levels post-vaccination may be the inclusion of poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) as an adjuvant component in the korv vaccine. pcep has been previously shown to induce a strong ifn-γ response and enhance antigenspecific immune response in mice following vaccination [ ] . several host restriction factors able to interfere with multiple stages of the viral life cycle have been identified in humans and animals [ ] . our study investigated the expression of four host restriction factors in koalas harbouring endogenous korv: bst , isg , rsad and trim . all four genes were found to be expressed at quantifiable levels. surprisingly, bst , isg and rsad were expressed at relatively high levels, suggesting that these restriction factors may have biological importance in koalas harbouring endogenous korv. although there was not a vaccine-induced up-regulation in these host restriction genes, the detection and investigation of these genes nonetheless present a new line of research into innate antiviral mechanisms for koalas. the role of korv in modulating the cd :cd ratio in koalas has been previously investigated. a study of captive koalas harbouring endogenous korv reported a cd :cd expression median ratio of . (range: . - . ) [ ] . surprisingly, all koalas in the current study had very low levels of cd mrna expression when compared to cd β mrna expression. this observation was not improved by vaccination. when compared to the normal range in humans ( . - . ) [ ] , the levels observed in this work suggest an abnormally low cd expression in koalas. unfortunately, the lack of koala-specific reagents prevented further investigation into whether the reduced cd mrna expression directly translated into fewer cd t cells. however, a positive association between cd molecule and cd mrna expression has been previously shown in human studies [ ] . as such, these results could suggest an ongoing korv-associated immunosuppression, possibly through the preferential loss of cd t cells in korv-infected koalas, similar to what is seen in cats infected with feline leukemia virus and feline immunodeficiency virus [ ] . finally, il- was by far the most expressed cytokine detected in tested koalas. increased il- expression has been implicated in several pathologies including leukemia [ ] . high levels of il- is also associated with progression to disease in people infected with hiv- , when compared to those who were able to maintain natural control of the infection [ , ] . the extremely high level of il- seen in these korv infected koalas is highly interesting, as it may be an important biomarker for underlying conditions that may be korv-associated. in these endogenously infected koalas, korv vaccination led to a small but significant decrease in il- mrna expression. a recent study showed that reduction in il- levels was required for chlamydia clearance in korvinfected koalas [ ] . if further studies can conclusively link il- to koala pathologies, then the role of il- inhibitors, such as through vaccination, should be further explored. this work described increased ifn-γ mrna expression following korv vaccination in koalas harbouring endogenous korv. a very high expression level of il- mrna prior to vaccination, followed by a slight but significant decrease post-vaccination, was also observed. finally, this work provides new insight into possible mechanisms for korv-associated immunosuppression in korv infected koalas. korv: koala retrovirus; il: interleukin; ifn: interferon. retroviral invasion of the koala genome long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline helping koalas battle disease-recent advances in chlamydia and koala retrovirus (korv) disease understanding and treatment in koalas altered immune parameters associated with koala retrovirus (korv) and chlamydial infection in free ranging victorian koalas (phascolarctos cinereus) altered immune cytokine expression associated with korv b infection and season in captive koalas an exogenous retrovirus isolated from koalas with malignant neoplasias in a us zoo therapeutic vaccination of koalas harbouring endogenous koala retrovirus (korv) improves antibody responses and reduces circulating viral load memory t cell proliferative responses and ifn-γ productivity sustain long-lasting efficacy of a cap-based pcv vaccine upon pcv natural infection and associated disease b cells responses and cytokine production are regulated by their immune microenvironment poly[di(sodium carboxylatoethylphenoxy)phosphazene] (pcep) is a potent enhancer of mixed th /th immune responses in mice immunized with influenza virus antigens restriction factors: from intrinsic viral restriction to shaping cellular immunity against hiv- expression profiles of the immune genes cd , cd β, ifnγ, il- , il- and il- in mitogen-stimulated koala lymphocytes (phascolarctos cinereus) by qrt-pcr imbalance in the game of t cells: what can the cd /cd t-cell ratio tell us about hiv and health? plospathog correlation between the expression of cd and the level of cd mrna in human b-cell lines clinical aspects of feline retroviruses: a review plasma interleukin level predicts for survival in chronic lymphocytic leukaemia host factor predictors in long-term nonprogressors hiv- infected with distinct viral clades il- alterations in hiv- infected children with disease progression antibiotic treatment of chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the many groups that have significantly supported the overall koala vaccine development work, including the departments of transport and main roads, and environment and heritage protection of the queensland government, the moreton bay rail project team, moreton bay regional council, city of gold coast, redland city council, lone pine koala sanctuary, endeavour veterinary ecology, koala action inc., friends of koala (lismore), australia zoo wildlife hospital, staff and volunteers at the adelaide koala and wildlife hospital, as well as all koala rescue groups, zoos south australia, and vaccine and infectious diseases organization (vido), canada. we would also like to thank dr nic west and dr jelena vider (systems biology and data science, griffith university, australia) for help with nanostring experimentation. this work was financially supported by the australian research council linkage grant lp received by pt. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the data that support the findings of this study are available from the authors on request. all animal work reported in this study was approved by university of the sunshine coast animal ethics committee (ana ). this study was conducted in compliance with approved institutional guidelines. not applicable. the authors declare no competing interests.received: july accepted: october key: cord- -mwzz db authors: lu, guilan; gonzalez, richard; guo, li; wu, chao; wu, jiang; vernet, guy; paranhos-baccalà, gláucia; wang, jianwei; hung, tao title: large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing, china date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: mwzz db background: human metapneumovirus (hmpv), a recently identified virus, causes acute respiratory tract infections (artis) in infants and children. however, studies on the seroepidemeology of hmpv are very limited in china. to assess the seroprevalence of hmpv infection in china, we tested a total of , serum specimens for the presence of anti-hmpv igg antibody in children and adults free of acute respiratory illness in beijing, china by using hmpv nucleocapsid (n) protein as an antigen. as a control, we used the human serum antibody against the n protein of human respiratory syncytial virus (hrsv), the most important viral agent responsible for aris in children. results: the seropositive rate for hmpv increased steadily with age from % at - mo to % at age . however, the rate dropped slightly between mo and yr of age. the seropositive rate for hrsv also increased steadily with age from % at - mo to % at age . in children aged six months to six years, the seropositive rates for the anti-hrsv igg antibody were significantly higher than those for hmpv. additionally, igg antibody titers to hmpv and hrsv were significantly higher in adults than in young children. consistent with the seropositive rates, the geometric mean titer of anti-hmpv igg antibody was lower than that of anti-hrsv igg antibody in children aged six months to six years. conclusions: our results indicate that similar to hrsv, exposure to hmpv is ubiquitous in the beijing population. however, the seroprevalence of anti-hmpv igg antibody is lower than that of hrsv in children between six months and six years old, which suggests a different number of repeat infections or a different response to infections. human metapneumovirus (hmpv), thought to belong to the metapneumovirus genus of the pneumovirinae subfamily, is a recently identified human respiratory pathogen first isolated from hospitalized children with acute respiratory infections (aris) in the netherlands [ ] . the viral genome, clinical manifestations, and epidemiology associated with hmpv are similar to those of human respiratory syncytial virus (hrsv), which was identified in and is the most important viral agent responsible for aris in children [ ] [ ] [ ] . since its initial identification, hmpv infections have been reported worldwide. however, fluctuating incidence of its infection has been reported by groups from different areas, varying from . % to % in respiratory tract samples from patients with aris [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . seroepidemiological investigations also showed that the prevalence of hmpv may differ between geographical locations. for example, in the netherlands, virtually all children have been exposed to hmpv by the age of five, demonstrating that hmpv infection is common in childhood [ ] . in canada, an elisa-based test using recombinant nucleocapsid protein (n protein) of hmpv produced by baculovirus revealed that more than % of patients over years of age tested seropositive for hmpv [ ] . in china, however, studies on the seroepidemeology of hmpv have been limited, and it is unclear what percentage of the population in different age groups have been infected with the virus. the n protein, about amino acids in length, is encoded by the n gene of hmpv genome. the hmpv n protein is abundantly expressed during the early replication stage of the virus and can stimulate a sustained immune response [ ] . because the amino acid identity of hmpv n is highly conserved within and between the a and b subgroups of hmpv [ ] , it has been widely applied in the immunoassay of hmpv infection and in the investigation of seroprevalence of hmpv infections [ , ] . hmpv n protein shares - % homology of amino acid sequence with hrsv [ ] . as previous studies have not shown obvious cross-reactions in immunoassays such as elisa, the hrsv n protein has been used as a reference to evaluate the seroprevalence of hmpv [ , ] . to assess the seroprevalence of hmpv infection in china, we used hmpv n protein as an antigen to test serum samples for the presence of anti-hmpv igg antibody in children and adults free of acute respiratory illness in beijing, china. the igg antibody against n protein of hrsv was tested in parallel as a control. our results indicate that exposure to hmpv is ubiquitous in the beijing population. lower seropositive rates and geometric mean titer (gmt) of anti-hmpv igg were observed in children aged six months to six years when compared to hrsv. this may reflect the divergence of infection pattern between hmpv and hrsv in children. our data will inform the evaluation of the social and economic burden of hmpv infection and enable the development of medical or public health strategies to combat hmpv infection in the population. establishment of an elisa-based detection method for seroprevalence of hmpv and hrsv igg antibody both recombinant hmpv and hrsv n proteins were effectively expressed in e. coli bl (de ) as soluble proteins. recombinant hmpv n protein was then purified from bl (de ) cell lysates by ni-chelating chromatography, and recombinant hrsv n protein was purified by anion exchange chromatography followed by ni-chelating chromatography (data not shown). the purity of both recombinant n proteins was greater than %. the purified proteins were confirmed by western blot analysis using mouse anti- ×his tag monoclonal antibody as the primary antibody and irdye goat anti-mouse monoclonal antibody as the secondary antibody ( figure ). to optimize elisa conditions, we performed chessboard titration tests using the recombinant hmpv and hrsv n proteins. hmpv-positive and negative sera samples were identified by western blot analysis (figure a , left panel). the optimal antigen (hmpv n protein) concentration for elisa under our test conditions was determined to be . μg/ml for hmpv, because at a : serum dilution, the od nm value of positive sera was approximately . and the difference between the od nm value of positive and negative sera was greatest (figure a, right panel) . similarly, we determined the optimal assay conditions for hrsv n protein-elisa to be . μg/ml hrsv n protein at : serum dilution ( figure b ). based on these elisa conditions, we then determined the cut-off value for these assays. twenty-eight anti-hmpv igg negative and anti-hrsv igg negative sera samples were identified using western blot analysis of sera samples randomly collected from children under five years of age. the od nm of each negative sample was determined by elisa for hmpv n protein or hrsv n protein. the mean value of the od nm of the hmpv negative sera was . with a standard deviation (sd) of . . the mean value of the od nm of the hrsv negative sera was . with a sd of . . based on the formula: cut-off value = mean od nm of the negative sera + three fold sds [ , ] , the hmpv and hrsv positive cut-off values were then defined as . and . . a tested sample was scored as "positive" if its od nm value was above the cut-off value. the sensitivity of these two elisa methods was determined using purified murine igg against hmpv or hrsv n proteins. the limits of detection were . μg/ml igg and . μg/ml igg, respectively. to test the specificity of the elisa methods established in this study, the reactions of mouse sera against influenza virus a (subtypes h -h ), human coronaviruses ( e, hku and nl ), and polyomavirus jc against hmpv and hrsv n protein were evaluated. there was no obvious cross-reaction between those mouse antibodies and the hmpv or hrsv n proteins (data not shown). furthermore, we did not observe cross-reaction between the hrsv n protein and hmpv n protein (data not shown). for large-scale elisa screening, each of the tested serum samples was evaluated for the presence of anti-hmpv and anti-hrsv (n protein) antibodies. our analysis indicates that among the , samples, % ( / ) of subjects aged - mo, % ( / ) of subjects aged mo- yr, % ( / ) aged - yr, % ( / ) aged - yr, % ( / ) aged - yr, and % ( / ) aged - yr were seropositive for hmpv; whereas % ( / ) of subjects aged - mo, % ( / ) aged mo- yr, % ( / ) aged - yr, % ( / ) aged - yr, % ( / ) aged - yr, and % ( / ) aged - yr were seropositive for hrsv ( figure and table ). comparison between the hmpv and hrsvpositive sera by statistical analysis (χ tests) demonstrated that the seropositive rates of hmpv were significantly lower than those of hrsv in the age groups of six months to six years, whereas no significant difference was observed in the age groups of one to six months, or six years to years ( table ) . to characterize the seroprevalence of hmpv infection in the sample used in this study further, we analyzed the titers of anti-hmpv and anti-hrsv igg antibodies in our samples. twenty randomly selected seropositive samples in each age group were analyzed. the geometric mean titers (gmts) of anti-hmpv igg and anti-hrsv igg were significantly higher in the age group of > years than in the age group of ≤ years (for gmt of anti-hmpv igg, vs , p = . ; for anti-hrsv igg, vs , p = . , mann-whitney u test) ( figure ). consistent with the seropositive rates, the gmt of anti-hmpv igg ( ) was also lower than that of anti-hrsv igg ( ) in a younger group between six months and six years of age (mann-whitney u test, p = . ). in this age group, the proportion of specimens with a low anti-hmpv igg titer (≤ : ) was significantly higher than those with a low anti-hrsv igg titer ( . % vs . %, χ test, p = . ) ( figure ). notably, for individuals over six years of age, the proportion of specimens with a high anti-hmpv titer (≥ : ) increased and was comparable to those with a high anti-hrsv igg titer ( . % vs . %, χ test, p = . ) (figures and ). in this report, we show a high seroprevalence of anti-hmpv igg antibody in the beijing area. the , serum samples included in our study were randomly collected in , from individuals receiving routine physical examinations and who were free of recent respiratory infections. thus, our data should represent the incidence of hmpv infection in beijing. our results indicate that virtually all children over the age of six years have been exposed to hmpv infection. compared to reports from the netherlands and japan in which and samples were tested [ , ], our data were derived from a larger number of subjects. consistent with those two reports, our results suggest that hmpv infection is ubiquitous. however, our results showed a higher rate of hmpv infection in infants and younger children than reported in the netherlands or japan. we found that the seroprevalence of hmpv in children aged mo to yr was % compared to % in the netherlands and . % in japan. thus, our data suggests that primary infection of children by hmpv may occur much earlier in beijing than in the netherlands or japan. this discrepancy may be caused by differences in exposure to hmpv in different geographical regions, or by other factors such as social, environmental, or climate conditions. similarly, hrsv has a high seroprevalence rate in beijing. our data correlate with previous findings that seroprevalence increases with age and that by the age of three, over % of children are seropositive for hrsv [ ] . interestingly, our results demonstrate that the seropositive rate of hmpv in children six months to six years of age are significantly lower than seropositive rates of hrsv (p < . ), suggesting that hrsv infection occurs earlier than hmpv infection. our results showed that the seropositivity of hmpv decreases during the mo- year period. maternal antibodies may be responsible for higher hmpv seropositivity in individuals - months old. the seropositivity of hmpv decreases during the mo- year period as the level of the maternal antibody decreases. the antibody titers then increase with age, after repeat hmpv infection. notably, within the age group of mo to yr, the gmts of anti-hmpv igg were significantly lower than those of anti-hrsv igg. significantly more specimens had a low anti-hmpv igg (≤ : ) titer than had a low anti-hrsv igg titer. the reasons for this disparity are unclear. it is possible that infection by hmpv occurs later than infection by hrsv, thus leading to the delayed increase in the seropositive rate and antibody titer of hmpv [ ] . it is likely that less response to hmpv or more exposure to hrsv that would boost the antibody titer, results in a higher antibody titer against hrsv than against hmpv. however, large-scale investigations using samples collected from different geographic regions are necessary to distinguish the immunological responses to the two viruses during early human life. we did not find significant differences between the seroprevalence of hmpv and hrsv infections in children less than six months of age. this reflects high levels of maternal antibody against hmpv and hrsv, which may a hmpv b hrsv figure titers of anti-hmpv and anti-hrsv igg antibodies. twenty human serum samples that were seropositive for hmpv (a) or hrsv (b) were randomly selected from each age group, for a total of samples. elisa was performed using hmpv n protein or hrsv n protein as the antigen. titers are shown for the indicated age groups. horizontal bars indicate the gmts of antibodies against hmpv or hrsv. figure and ) . to determine the specificity of our elisa method, we tested for reactions between hmpv or hrsv n protein and murine antibodies against multiple viruses, including influenza a viruses (h -h ), human coronaviruses ( e, hku , nl ) and polyomavirus jc. we found no obvious reactivity in elisa assays. additionally, we did not detect a cross-reaction between hmpv n protein and hrsv n protein. these results indicate that the n protein-based elisa specifically detects both anti-hmpv and anti-hrsv antibodies. however, we need to further evaluate the specificity of the n protein-based elisa by using antibodies against other respiratory virus antigens. the sensitivity of the elisa method for detecting hmpv and hrsv was determined by using purified murine specific anti-hmpv or hrsv n igg, respectively. to our knowledge, limits of detection comparable to the sensitivity achieved by our methods have not been reported in other serological surveys of hmpv. repeated infections of an individual by hmpv and hrsv have been reported [ ] [ ] [ ] [ ] , suggesting that immunity against hmpv and hrsv is not solid, is transient, or is incomplete against heterologous strains. the high prevalence and high titers of hmpv and hrsv that we observed in adults may suggest that re-infection by hmpv and hrsv occurs throughout life [ , ] . our results suggest that similar to hrsv infection, hmpv infection is ubiquitous in the beijing population. however, the seroprevalence of the igg antibody against hmpv is lower than that against hrsv in children between six months and six years of age, which may reflect differences in infection pattern between the two viruses. our findings provide further information to aid the development of strategies to control and prevent hmpv infection. serum specimens were collected from , subjects, including children (from the beijing children's hospital) and adults (from the beijing blood center), during routine physical examinations in . all individuals were free of acute respiratory infection for at least three months prior to sampling. all samples were collected after obtaining informed consent either from the individuals or from the individual's guardians. the sera were separated immediately after collection, stored at - °c, and inactivated at °c for minutes before use. purified recombinant proteins were separated by % sds-page and transferred onto a nitrocellulose membrane, as previously described [ ] . the membrane was blocked for two hours in % non-fat milk. mouse anti-his monoclonal antibody (sigma, munich, germany) diluted : in non-fat milk or serum samples diluted : in non-fat milk were added, and membranes were incubated for one hour at room temperature. membranes were then washed three times with pbs- . % tween and incubated for one hour at room temperature with the secondary antibody, irdye goat antimouse monoclonal antibody (li-cor biosciences, nebraska, usa) or horseradish peroxidase-conjugated goat anti-human igg monoclonal antibody (sigma, munich, germany). subsequently, membranes were washed three times with pbs- . % tween, then developed using odyssey (li-cor biosciences) or a tetramethylbenzidine (tmb) substrate (thermo scientific, rockford, usa) according to the manufacturers' instructions. chessboard titration tests were conducted using positive and negative serum samples that were randomly selected. western blot analysis was used to determine the optimal concentration of the coated antigen and serum dilution. subsequently, -well plates (costar, bethesda, usa) were coated with μl of . μg/ml purified hmpv n proteins or . μg/ml purified hrsv n proteins, in . m sodium hydrogen carbonate buffer (ph . ). plates were incubated overnight at °c, then blocked with % bsa overnight at °c and washed three times with pbs- . % tween. subsequently, μl of a : dilution of serum specimens was added and incubated at °c for one hour. the plates were then washed six times with pbs- . % tween and incubated for one hour at °c with horseradish peroxidase-conjugated goat anti-human igg (sigma) diluted : , . the plates were washed again six times with the same solution, and antibodies were detected by adding μl substrate solutions a and b (wantai biotech corp. beijing, china) followed by incubation at °c for minutes. the reactions were terminated by adding μl of m h so . optical densities (od) were read at nm (od nm ). the average od values for the hmpv-negative human sera samples (n = ), shown to be negative by western blot analysis for the hmpv-n protein, were measured. the average od values of the hrsv-negative human sera samples (n = ), shown to be negative by western blot analysis for the hrsv-n protein, were also measured. the cut-off values were defined as the mean od of the negative sera plus three standard deviations [ , ] . samples with od nm values above the cut-off value were considered positive. to determine the specificity of our elisa method, we tested mouse sera against inflenza virus ha proteins (subtype h -h ), human coronavirus ( e, hku , nl ) n proteins, and polyomavirus jc vp protein for reaction against hmpv and hrsv proteins. samples of sera were tested in serial dilutions of two-fold, starting at a : dilution. to determine the sensitivity of elisa method established in this study, mice sera against hmpv n protein and hrsv n protein were purified using protein-g sepharose column (ge healthcare) and quantified using pierce ® bca protein assay kit (thermo scientific, rockford). the purified murine igg against hmpv or hrsv were tested in serial dilutions of two-fold (starting at as μg/ml concentration). in addition, randomly selected positive serum samples in each group were subjected to anti-hmpv and anti-hrsv titer assays. to determine the endpoints of antibody titers, titers were calculated as the highest dilution of a serum showing an od nm reading of two times the mean of the negative serum control (starting at a : ) [ ] . seropositive rates were evaluated using χ tests. mean antibody titers between children and adults positive for hmpv and hrsv exposure were analyzed using the mann-whitney u test. a p value ≤ . was considered statistically significant. peking union medical college (pumc) & chinese academy of medical sciences (cams) peking union medical college & chinese academy of medical sciences, beijing , china. fondation mérieux, lyon , 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and poultry from lethal h n avian influenza virus through adenovirus based immunization submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the beijing xicheng district cdc and beijing children's hospital for their assistance with the collection of sera specimens. this study is supported in part by the national major science and technology research projects for the control and prevention of major infectious diseases in china ( zx - ). authors' contributions gl, rg, and jw designed the study. gl, lg, and cw conducted the experiments. jw was in charge of the collection of sera specimens. gl, rg, gp, gv and jw wrote the manuscript, and gv, gp, jw and th revised the manuscript. all authors have read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -vfe lawl authors: riddell, shane; goldie, sarah; hill, andrew; eagles, debbie; drew, trevor w. title: the effect of temperature on persistence of sars-cov- on common surfaces date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: vfe lawl background: the rate at which covid- has spread throughout the globe has been alarming. while the role of fomite transmission is not yet fully understood, precise data on the environmental stability of sars-cov- is required to determine the risks of fomite transmission from contaminated surfaces. methods: this study measured the survival rates of infectious sars-cov- , suspended in a standard astm e matrix, on several common surface types. all experiments were carried out in the dark, to negate any effects of uv light. inoculated surfaces were incubated at °c, °c and °c and sampled at various time points. results: survival rates of sars-cov- were determined at different temperatures and d-values, z-values and half-life were calculated. we obtained half lives of between . and . days at °c, reducing to a few hours when temperature was elevated to °c. with initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to days at °c from common surfaces such as glass, stainless steel and both paper and polymer banknotes. conversely, infectious virus survived less than h at °c on some surfaces. conclusion: these findings demonstrate sars-cov- can remain infectious for significantly longer time periods than generally considered possible. these results could be used to inform improved risk mitigation procedures to prevent the fomite spread of covid- . the world health organization (who) declared sars-cov- a pandemic on th march and as at the th august , there have been over . million confirmed cases with more than , reported deaths from sars-cov- [ ] . the transmission of sars-cov- appears to be primarily via aerosols [ ] [ ] [ ] and recent studies have shown that sars-cov- is able to remain infectious in airborne particles for greater than h [ , ] . the role of fomites in the current pandemic is yet to be fully determined, although they have been suggested as a potential mode of transmission [ ] also reflected by the strong focus on hand-washing by who and national control schemes. broadly, viruses have been shown to be readily transferred between contaminated skin and a fomite surface [ ] , with high contact surfaces such as touchscreens on mobile phones, bank atms, airport check-in kiosks and supermarket self-serve kiosks all acting as fomites for the transmission of viruses [ ] . fomite transmission has previously been shown to be a highly efficient procedure, with transmission efficiencies of % for both fomite to hand and fingertip to mouth transfer for bacteria and phages [ ] . with the high efficiency of fomite transfer, the persistence of sars-cov- on environmental surfaces is therefore a critical factor when considering the potential for fomite transmission for this virus. currently, there are conflicting reports on the survivability of sars-cov- , with data ranging from to days at room temperature for a single surface type, stainless steel open access *correspondence: shane.riddell@csiro.au commonwealth scientific and industrial research organisation (csiro), australian centre for disease preparedness, geelong, vic, australia [ , ] . this study aims to provide environmental stability data for sars-cov- under controlled temperature and humidity conditions for a range of common surfaces. the sars-cov- isolate (betacoronavirus/australia/ sa / ) used in this study was kindly supplied by the peter doherty institute (victoria, australia) on behalf of south australian health (south australia). the virus was passaged four times through vero e cells (atcc crl- ) in dulbecco's modified eagle medium (dmem) supplemented with penicillin, streptomycin, fungizone and % fetal calf serum and pelleted via ultracentrifugation at , ×g for min. the virus was resuspended in phosphate buffered saline (pbs) with % bovine serum albumin (bsa) and stored at − °c. the virus stock was titrated on vero e cells and the tcid was determined to be . × /ml by the spearman-karber method [ , ] . all work with infectious sars-cov- was conducted in the high containment laboratory (biosafety level ) at the australian centre for disease preparedness. australian polymer bank notes, de-monetised paper bank notes and common surfaces including brushed stainless steel, glass, vinyl and cotton cloth were used as substrates in this study. both polymer and paper banknotes were included in the study to gather information on the possible roles of note based currency in general for the potential for fomite transmission. stainless steel is used in kitchen areas and public facilities and is the substrate used in some disinfectant testing standards [ , ] . glass was chosen due to its prevalence in public areas, including hospital waiting rooms, public transport windows and shopping centres, and high contact surfaces such as mobile phone screens, atms and self-serve check-out machines. vinyl is a common substrate used in social settings, tables, flooring, grab handles on public transport, as well as mobile phone screen protector material. cotton was chosen as a porous substrate, often found in clothing, bedding and household fabrics. all surfaces were prepared by cutting into approx. - . cm coupons, non-porous surfaces were disinfected prior to use by washing in a mild detergent (beckman ), rinsing in distilled water and then immersing in % v/v ethanol. paper bank notes (in very good condition) were heated in a dry oven to °c for h to reduce bacterial/viral contamination. the % cotton cloth was steam sterilised prior to use. following preparation, all surfaces were placed into a petri dish and allowed to dry in a class ii biological safety cabinet (bscii) at room temperature and humidity prior to inoculation. stock virus was diluted in a defined organic matrix, consisting of bovine serum albumin (bsa), mucin and tryptone, following international standard astm e [ ] , designed to mimic the composition of body secretions. briefly, µl of virus stock was added to µl of a solution consisting of . mg/ml bsa, . mg/ml tryptone and . mg/ml mucin. ten microlitres of the resulting suspension (final concentration of . × / µl) was inoculated onto the centre of the coupon and allowed to dry in a bscii for h. once dry, the coupons were placed into a humidified climate chamber (memmert hpp ) for specified time points. samples were incubated in the dark to limit any effect light might have on viral decay. a single humidity set point ( % relative humidity) was maintained for each of three separate temperature experiments ( °c, °c, °c). for the °c and °c temperature experiments, three replicates of each surface type were inoculated and sampled at the following time points; h, day, days, days, days, days and days post inoculation. for the °c experiment, triplicate samples were inoculated for the following time points; h, day, days, days, days, and days. for non-porous surfaces, for each replicate, virus was eluted in × µl volumes of dmem with repeated pipetting then titrated individually, in quadruplicate wells on a -well plate. for recovery from cotton cloth, inoculated swatches of the cloth were individually submersed in µl dmem and pipetted repeatedly for at least min before µl of the recovered eluent from each swatch was titrated separately, in quadruplicate. suspensions of vero e cells ( × /ml) were added to the wells and the plates were incubated for days at °c with % co . wells were scored for the presence of cytopathic effect and titres calculated using the spearman-karber method. data analysis (regression analysis) and graphical representations were performed using graphpad prism (version ). decimal reduction time (d value-time at which there was a one log/ % reduction in titre) was calculated using z-values (temperature change required to achieve a tenfold (i.e. log ) change in the d value) was calculated d = t logn − logn f by plotting log d values against temperature. calculated using: the half-life of each surface was calculated using; at °c, infectious sars-cov- virus was still detectable after days post inoculation, for all non-porous surfaces tested (glass, polymer note, stainless steel, vinyl and paper notes). the recovery of sars-cov- on porous material (cotton cloth) was reduced compared with most non-porous surfaces, with no infectious virus recovered past day post inoculation. the majority of virus reduction on cotton occurred very soon after application of virus, suggesting an immediate adsorption effect. the calculated d values for surfaces at °c ranged from . days for cotton to . days for paper notes and are shown in table . at °c, infectious virus was recoverable for days from stainless steel, polymer notes and glass, and days for vinyl and cotton cloth. for paper notes, infectious virus was detected for days, although there was less than log of virus recovered for both day and day time points. the d values for surfaces at °c ranged from . days for vinyl to . days for paper notes (table ) . at °c, virus recovery was significantly reduced compared to both °c and °c experiments. infectious sars-cov- was not recovered past h for cotton cloth and h for all remaining surfaces tested. greater than z = (t − t )/ logd − logd t / = log k -log reduction ( . % reduction from starting titre) was observed in less than h at °c on all surfaces. the d values for surfaces at °c have been converted to hours as they were all less than day, values ranged from h for polymer notes to . h for vinyl (table ) . for each temperature and substrate material, the mean titre from three replicates of recovered virus was plotted against time, with standard deviations included. linear regression was used to calculate a line of best fit. plots showing virus survival on each substrate at the three temperatures investigated are shown in fig. . plots presenting this data grouping all substrates at each of the three temperatures are given in fig. . calculated d-value, half life and z-value are presented in table . an additional table containing average titre and standard deviation for all substrates, time points and temperatures is available (see additional file ). while the primary spread of sars-cov- appears to be via aerosols and respiratory droplets, fomites may also be an important contributor in transmission of the virus. fomite transmission has been demonstrated as an important factor in the spread other coronaviruses such as porcine epidemic diarrhea virus [ ] , as well as being suspected for middle east respiratory syndrome coronavirus [ ] , human coronavirus e and oc [ ] and sars-cov- [ ] . this study utilised a virus concentration of . × / ml diluted into a standard solution which mimics body fluid composition (final concentration of . × / µl inoculum), which equates to a cycle threshold (ct) value of . , . and . for n gene, e gene and rdrp gene real time rt-pcr, respectively (unpublished data). previous studies have shown some patients with high viral loads have recorded ct values of between and [ ] [ ] [ ] . van doremalen et al. [ ] described their test material ( tcid /ml) as having a ct of - , which compared similarly to cts reported from clinical patients [ , ] . while the titre of virus utilised in this study is high it represents a plausible amount of virus that may be deposited on a surface. the present study has demonstrated that in controlled conditions, sars-cov- at a starting viral load and in a fluid matrix equivalent to that typically excreted by infected patients, remains viable for at least days when dried onto non-porous surfaces at °c and % relative humidity. research on the original sars virus also showed recovery of infectious virus when dried on plastic for up to days at room temperature and - % rh [ ] . recent data published on sars-cov- survivability on hospital ppe observed viable virus up to days post inoculation on both plastic and n mask material when held at room temperature [ ] , correlating with the data presented in this study. the persistence of sars-cov- on surfaces presented here and from kasloff et al. [ ] demonstrate significantly longer time points than previously published data for sars-cov- [ , ] . these earlier studies reported recovery of infectious sars-cov- up to days post inoculation and days on non-porous surfaces, respectively. the titre of virus used in this study is at least logs higher than used in the paper by van doremalen et al. [ ] , which may account for the longer survivability. work by lai et al. has shown that stability of sars virus was enhanced with higher concentrations [ ] . temperature and humidity are both critical factors in viral survivability with an increase in either being detrimental to virus survival [ , , ] . survivability on stainless steel coupons for transmissible gastroenteritis virus and murine hepatitis virus (both coronaviruses) was reduced with higher humidity's and temperature [ ] and survivability of middle east respiratory syndrome coronavirus also followed a similar pattern [ ] . the higher humidity of ~ % rh used by chin et al. [ ] may explain the shorter persistence of virus when compared to the data presented here. sars-cov- has been shown to be rapidly inactivated under simulated sunlight [ , ] . to remove any potential decay by light sources, inoculated coupons were held in the dark for the duration of the experiment. decimal reduction (d value; the timetaken to reduce the titre by log) for sars-cov- at °c and %rh ranged from . to . days (average . ) for all surfaces tested. this data is significantly longer than modelling predications performed by guillier et al. [ ] . the data presented here was performed under controlled conditions with fixed temperatures, relative humidity, suspension matrix and in the absence of light, which may explain the enhanced survivability observed in this study. the generation of z values at different temperatures also allows for extrapolation of d values for each surface at other temperatures. the z value represents the temperature change required to alter the d value by log. for stainless steel, the d value was determined to be . days at °c, and the z value of . °c, therefore if the temperature was to drop by . °c from °c (i.e. to . °c), then the d value would increase from . days to over days. this data could therefore provide a reasonable explanation for the outbreaks of covid- surrounding meat processing and cold storage facilities. the data also supports the findings of a recent publication on survival of sars-cov- on fresh and frozen food [ ] . stainless steel is a common surface for study of viral stability, and has been used to study the persistence on a number of viruses such as ebola virus, hepatitis virus, influenza a and coronaviruses [ , [ ] [ ] [ ] [ ] . this study demonstrates that sars-cov- is extremely stable on stainless steel surfaces at room temperature (> days at °c/ %rh) however, is less stable at elevated temperatures ( days at °c and < h at °c). recovery of infectious virus on stainless steel has been observed for murine hepatitis virus and transmissible gastroenteritis virus for up to days albeit at a lower humidity %rh [ ] . interestingly, the same study showed survivability at °c and %rh was significantly less ( - days), further suggesting the humidity may play a significant role in virus survival. the persistence of virus on both paper and polymer currency is of particular significance, considering the frequency of circulation and the potential for transfer of viable virus both between individuals and geographic locations. while other studies have shown that paper notes harbour more pathogens than polymer notes [ ] , this data demonstrates that sars-cov- persists on both paper notes and polymer notes to at least days at °c, albeit with a faster rate of inactivation on polymer notes. data presented in this study for banknotes is significantly longer than reported for other respiratory viruses such as influenza a (h n ) which demonstrated survival up to days at room temperature [ ] . it is also noted that prior to sars-cov- being declared a pandemic, china had commenced decontamination of its paper based currency, suggesting concerns over transmission via paper banknotes existed at the time [ , ] . the united states and south korea have also quarantined bank notes as a result of the pandemic [ , ] . it is important to note that after days, infectious sars-cov- was also recovered from stainless steel, vinyl and glass, suggesting survivability on paper or polymer banknotes was not very different from the other non-porous surfaces studied. the persistence on glass is an important finding, given that touchscreen devices such as mobile phones, bank atms, supermarket self-serve checkouts and airport check-in kiosks are high touch surfaces which may not be regularly cleaned and therefore pose a transmission risk of sars-cov- . it has been demonstrated that mobile phones can harbour pathogens responsible for nosocomial transmission [ ] , and unlike hands, are not regularly cleaned [ ] . the data presented in this study correlates well with previously published data for influenza a (h n ) which recovered infectious virus up to days at °c and days at °c [ ] . the persistence of sars-cov- on glass and vinyl (both common screen and screen protector materials, suggest that touchscreen devices may provide a potential source of transmission, and should regularly be disinfected especially in multi-user environments. the persistence of both sars and sars-cov- on cotton has been demonstrated to be significantly shorter than on non-porous surfaces [ , ] . the data presented here also shows a significant decrease in titre of recovered virus after just h drying at room temperature ( °c) the amount of virus recovered from cotton swatches was approximately % less than for comparable virus recovery time points for non-porous material. to verify the reduced recovery on cotton, virus was eluted min after depositing on the cotton, as well as h, the titre of recovered virus after min was similar to that of non-porous surfaces (data not shown) suggesting the process of drying down was a significant factor for cotton material but not from the non-porous surfaces. recovery of virus from porous substrates is also likely to be reduced compared to nonporous substrates due to adherence of the virus to the fabric fibres. when the rate of viral inactivation is considered over time rather than the gross reduction from the initial inoculum there is a more subtle difference from the non-porous surfaces. the d values for cotton at °c, when compared other materials, are not significantly different from other substrates (eg. . days for cotton vs. . days for vinyl), and the slopes of the line which suggests the decay rate of virus is similar across substrates. this study also demonstrates significantly longer survival times on cotton ( days) than previous reported [ , ] . this difference could be due to differences in the types of cotton material used, the current study used % cotton cloth, while previous studies used either a cotton gown or cotton t-shirt. the data presented in this study demonstrates that infectious sars-cov- can be recovered from nonporous surfaces for at least days at ambient temperature and humidity ( °c and % rh). increasing the temperature while maintaining humidity drastically reduced the survivability of the virus to as little as h at °c. the persistence of sars-cov- demonstrated in this study is pertinent to the public health and transport sectors. this data should be considered in strategies 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phones represent a pathway for microbial transmission: a scoping review publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations virus strain used in this study was generously provided by sa health through the victorian infectious disease research laboratory, peter doherty institute, melbourne, victoria. supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file . authors' contributions sr-conceptualisation, data curation, formal analysis, investigation, methodology, writing-original draft. sg-conceptualisation, data curation, formal analysis, investigation, methodology, writing-review and editing. ah-conceptualisation, data curation, formal analysis, writing-review and editing, supervision. de-conceptualisation, writing-review and editing, funding acquisition. td-conceptualisation, analysis methodology, writing-review and editing. all authors read and approved the final manuscript. funding for the climate chambers utilised in this study was provided by a sars-cov- collaboration grant from the defence, science and technology group (australia). all data generated or analysed during the study is included in the additional file . not applicable. not applicable. the authors declare that they have no competing interests.received: august accepted: september key: cord- -so xl authors: ebert, gregor; paradkar, prasad n.; londrigan, sarah l. title: virology downunder, a meeting commentary from the lorne infection and immunity conference, australia date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: so xl the aim of this article is to summarise the virology content presented at the th lorne infection and immunity conference, australia, in february . the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the lorne infection and immunity conference is one of five scientific meetings held during each month of february in seaside town of lorne, on the great ocean road in victoria (australia). the specific aim of the meeting is to bring together basic, clinical and translational researchers -those who examine microbes and their impact on the innate or adaptive immune response, researchers who study the mechanisms that regulate immune responses, and those who apply this knowledge to preventing and treating infectious and inflammatory diseases. was the th lorne infection and immunity conference, convened by heidi drummer (burnet institute, melbourne, australia) and paul hertzog (hudson institute of medical research, melbourne, australia). the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the 'infection and inflammation' session of the meeting was opened with a well-received presentation by linfa wang (duke-nus medical school, singapore) entitled 'holy immune balance, batman', about the highly adapted immune response of bats to viral infections. bats have been characterized as an important reservoir of various zoonotic viruses including nipah, sars, mers, marburg and ebola viruses [ ] , but remarkably are able to live asymptomatically with otherwise potentially lethal viruses [ ] . wang and colleagues are interested in understanding the underlying immune mediated regulatory mechanisms that facilitate a highly effective balance between viral defense and tolerance in bats as viral hosts. during their evolutionary adaption to effective flight, bats not only developed elevated levels of basal alertness reflected in increased metabolic heart rate and body temperature, they also seem to have evolved a highly adapted immune defense against viral infections [ ] . remarkably, bats have increased tolerance to viral infections by exhibiting higher basal levels of innate defence regulators but also a dampened innate immune response upon infection, substitutional to increased responsiveness to viral pathogens [ ] . the bat innate immune response appears to be 'pre-activated' with higher basal levels of type i interferon expression, in contrast to humans, who are very quick responders to viral infections, but require a lot more dampening of their immune signals afterwards to get back to basal levels. current research also shows the absence of any aim mediated inflammasome activation [ ] , dampened nlrp mediated inflammasome activation [ ] and dampened sting activation [ ] in bats upon infection, which are all mediators of a robust type i interferon response. overall, wang et al. demonstrated that bats' response to stress in form of viral infections is more targeted and thus potentially more effective by numerous adaptions and modifications of the innate immune system. in 'viruses and their hosts' session, vinod sundaramoorthy (csiro-australian animal health laboratories) discussed a novel defence mechanism in neurons against rabies virus (rabv). rabv is a neurotropic virus, which causes tens of thousands of deaths every year, despite available vaccines [ ] . endemic dog rabies results in an ongoing risk to humans in many resource-limited countries, whereas rabies in wildlife is important in north america and europe [ ] . requirement for an uninterrupted vaccine cold chain and the high cost of the immunoglobulin component of rabies prophylaxis therapies substantiate the unmet need for novel rabv-specific antivirals [ ] . furthermore, the pathogenesis of rabv relating to viral replication in neurons is not fully understood [ ] . although most of the infection with rabv manifests as the 'furious' form, where virus silently spreads through the neuronal axon without any damage, in % of cases it can cause axonal damage in peripheral neurons leading to paralysis [ ] . using a new in vitro microfluidics model for studying synaptically connected neurons, sudaramoorthy investigated the pathogenesis of different strains of rabies virus, showing distinct mechanisms at play in determining disease outcomes for each strain. future efforts to define a key molecular determinant in the viral replication pathway in neurons may provide a novel therapeutic target. in , an association between zika virus (zikv) infection during pregnancy as a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn, was made [ ] . as such, there has been a plethora of recent zikv research focused on understanding the pathogenesis of disease, as well as immunity to the virus and the development of vaccines and effective therapeutics. novel findings pertaining to all of these areas were presented throughout the meeting. developments in understanding the structure of zikv particles was showcased by shee-mei lok (duke-nus, singapore). lok and colleagues have previously solved a thermally stable . Å resolution cryo-electron microscopy structure of zikv [ ] , and were able to now show high resolution structures of zikv at various stages of viral assembly. specifically, the organisation of the envelope protein of the virus and changes during assembly and maturation were presented. previously, lok's lab has also published structures of mature and immature dengue virus, providing insight into the viral maturation process [ ] . the research from lok and colleagues will have future implications in designing novel therapeutics and vaccines for zikv. efforts to develop a zikv vaccine using virus like particles were presented by julio carrera, working with researchers at the monash university and the university of melbourne. in the 'pathogenesis and prevention of infection' session, rosa coldbeck-shackley working with michael beard at the university of adelaide, australia, and also colleagues at the hudson institute, presented findings on the importance of interferon-epsilon (ifn-ɛ) in the innate immune response to zikv infection. ifn-ɛ is a novel type i ifn, encoded within the type i ifn locus in mice and humans, whose function has recently been characterized [ ] . like other type i ifns, it acts via ifn-α receptors and , activating interferon stimulated genes (isgs). however, ifn-ɛ is preferentially expressed by epithelial cells of the female reproductive tract in both mice and humans, and in contrast to viral induced type i ifn expression, ifn-ɛ is hormonally regulated. other than mosquito-borne transmission, zikv is also sexually transmitted [ ] . this makes the research presented by coldbeck-shackley significant, and concurs with previous studies where ifn-ɛ-deficient mice were more susceptible to infection with sexually transmitted pathogens [ ] . thus, ifn-ε appears to be a potent antipathogen and immunoregulatory cytokine that may be important in combating sexually transmitted infections that represent a major global health and socioeconomic burden. also in the 'pathogenesis and prevention of infection' session, allison abendroth (university of sydney) presented 'disarming the killer: targeting of natural killer cells by varicella zoster virus'. varicella zoster virus (vzv), is known to infect numerous immune cell types such as t-cells and dendritic cells [ ] . moreover, the virus is able to modulate and manipulate innate and adaptive immune responses to infection to its replicative benefit. vzv infection of dendritic cells dampens type i ifn responses [ ] and leads to evasion of cd t cell recognition via downregulation of mhc class i expression [ ] . in addition, vzv mediated delay in immune responses to infection facilitates establishment of initial primary infection and lifelong latency in neurons. most recently, abendroth and colleagues found that vzv also productively infects natural killer (nk) cells and that nk cells effectively transmit infection to other permissive cell types [ ] . the group is now trying to understand, how nk cells, a cell type that normally demonstrates very effective direct and indirect antiviral capacity, is manipulated by vzv infection, leading to impaired cytotoxicity and cytokine responses upon infection and facilitating infection and spread of the virus. led by the observation that patients with impaired nk cell functionality are highly susceptible to severe and life threatening vzv infection, abendroth et al. found limited nk cell activation and efficacy upon vzv infection of target cells and characterised differential modulation of ligands normally recognised by activated nk cells [ ] . the exact underlying mechanisms, how vzv infection prevents the release of proinflammatory cytokines during infection of nk cells to impair their general function and whether correlations can be drawn to vzv infection of monocytes and macrophages [ ] , is currently under their investigation, abendroth stated. a key highlight at the conclusion of the meeting was a presentation from the victorian infection and immunity network young investigator prize winner, simone park (the university of melbourne). park, working alongside thomas gebhardt and laura mackay, has recently published seminal research investigating skin tissue-resident memory t cells (trm cells). specifically, park has demonstrated how these cells contribute to antiviral immune memory in peripheral tissues, using a herpes simplex model of infection [ ] . using an epicutaneous melanoma model, park and colleagues also demonstrate that trm cells play protective role in tumor surveillance [ ] , which has important implications for advancing anticancer immunotherapies. the organisers are looking forward to celebrating the th lorne infection and immunity meeting in and invite all researchers with an interest in infectious and inflammatory diseases and associated immune responses to participate. for more information: http://www.lor neinfectionimmunity.org/. viruses in bats and potential spillover to animals and humans 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provide insight into the maturation process interferon-epsilon protects the female reproductive tract from viral and bacterial infection leparc-goffart i. evidence of sexual transmission of zika virus varicella-zoster virus infection of human dendritic cells and transmission to t cells: implications for virus dissemination in the host impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection varicella-zoster virus productively infects mature dendritic cells and alters their immune function varicella zoster virus productively infects human natural killer cells and manipulates phenotype varicella -zoster virus and herpes simplex virus differentially modulate nkg d ligand expression during productive infection infection and functional modulation of human monocytes and macrophages by varicella-zoster virus local proliferation maintains a stable pool of tissue-resident memory t cells after antiviral recall responses tissue-resident memory cd (+) t cells promote melanoma-immune equilibrium in skin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions ge, pp and sl all contributed equally in the writing of this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - fp u authors: al-siyabi, turkiya; binkhamis, khalifa; wilcox, melanie; wong, sallene; pabbaraju, kanti; tellier, raymond; hatchette, todd f; leblanc, jason j title: a cost effective real-time pcr for the detection of adenovirus from viral swabs date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: fp u compared to traditional testing strategies, nucleic acid amplification tests such as real-time pcr offer many advantages for the detection of human adenoviruses. however, commercial assays are expensive and cost prohibitive for many clinical laboratories. to overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time pcr. in swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral dna obtained from a commercial nucleic acid extraction. in addition, the in-house real-time pcr outperformed traditional testing strategies using virus culture, with sensitivities of % and . %, respectively. overall, the combination of homogenization and heat treatment with a sensitive in-house real-time pcr provides accurate results at a cost comparable to viral culture. human adenoviruses (hadv) are ubiquitous dna viruses that cause a wide spectrum of illness [ ] . the majority of hadvs cause mild and self-limiting respiratory tract infections, gastroenteritis or conjunctivitis; however, more severe disease can occur such as kerato-conjunctivitis, pneumonitis, and disseminated disease in the immunodeficient host [ ] [ ] [ ] [ ] . hadv is increasingly being recognized as a significant viral pathogen, particularly in immunocompromized patients where accurate and timely diagnosis can play an integral part of management [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hadv diagnosis can be achieved using virus culture, antigen-based methods (immunofluorescence, enzyme immunoassays or immunochromatography), or nucleic acid amplification tests (naats). for respiratory viruses, naats are well established as the most sensitive methods for detection and have become front-line diagnostic procedures [ , [ ] [ ] [ ] [ ] [ ] [ ] . most commercially available naats are highly multiplexed assays and enable simultaneous detection of several respiratory pathogens; however, their poor performance for detecting hadv emphasizes the need for single target detection [ , , ] . adenovirusspecific naats have been challenged by the diversity of hadv species, which now include more than different types [ , ] . commercial qualitative and quantitative naats are available for the detection of all hadv species and most types, yet these are cost prohibitive for many laboratories. "in-house" real-time pcr assays are relatively inexpensive alternatives to commercial naats that provide rapid and accurate results [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . wong and collaborators [ ] developed an in-house real-time pcr assay that has been designed for the detection of all hadv species. it has been extensively validated using a variety of clinical specimens [ , ] . in addition to the pcr reaction itself, extraction of nucleic acids prior to pcr is also a substantial contributor to cost. recently, a crude mechanical lysis using silica glass beads (i.e. homogenization) and heat treatment was shown to recover herpes simplex virus dna from swabs submitted in universal transport media (utm) [ , ] . while defying the traditional paradigm of specimen processing for molecular testing, homogenization with heat treatment was shown to be a cost effective alternative to nucleic acid extraction. this study evaluated whether the combination of homogenization and heat treatment with an in-house real-time pcr would be a cost effective strategy for the detection of hadv from viral swabs transported in utm. in patients suspected of respiratory or conjunctivitis, flocked nasopharyngeal or ocular swabs, respectively, were submitted for adenovirus detection. swabs were collected by clinicians at the capital health district authority (cdha) and were submitted to the cdha microbiology laboratory (halifax, ns, canada) between april and march . the swabs were transported in ml of utm (copan diagnostics inc., murrieta, ca) and kept at °c for no more than hours prior to processing. viral cultures were performed as part of routine diagnostic testing by experienced technologists. following virus culture, specimens were transferred in aliquots into cryotubes (without any identifiable patient information) and the anonymized specimen tubes were archived at − °c for retrospective molecular analyses. twentyseven virus culture-positive specimens and virus culture-negative specimens were randomly selected and tested for the presence of hadv using a well established in-house real-time pcr assay [ ] following recovery of viral dna was recovered by homogenization with heat treatment or automated nucleic acid extraction. the world medical association (wma) declaration of helsinki is a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data. since the purpose of this clinical validation was quality improvement of the laboratory detection of adenovirus and relied exclusively on anonymous human biological materials that did not use or generate identifiable patient information, research ethics board (reb) review was not required based on chapter , article . of the tri-council policy statement: ethical conduct for research involving humans ( nd edition). viral cultures were performed as part of routine diagnostic testing by experienced technologists in the cdha microbiology laboratory (halifax, ns, canada). briefly, μl of specimen was inoculated onto cultured a cells (atcc ccl- ), incubated at °c in a % co atmosphere, and monitored daily for the presence of characteristic cytopathic effect (cpe) [ ] . if cpe was observed, cells were fixed with acetone and stained using specific fluorescein isothiocyanate (fitc)-labeled monoclonal antibodies in the d ultra dfa reagent kit (diagnostic hybrids, athens, oh). in absence of cpe, cells were fluid changed on day and incubated for an additional days. on day , the culture was discontinued and a terminal stain was performed. a cells were propagated in nutrient mixture f- ham with lglutamine (sigma-aldrich canada ltd., oakville, on) supplemented with % fetal calf serum (hyclone, thermo fisher scientific, ottawa, on), μg/ml amphotericin b (sigma-aldrich), μg/ml ampicillin (novapharm ltd, toronto, on), and mg/ml vancomycin (sigma-aldrich). for quantification, -fold dilutions of hadv-c, type (strain tonsil , atcc vr- ) were inoculated onto -well plates in volumes of μl. cells were maintained as described above and after days were subjected to direct immunofluorescence (dfa) to determine the % tissue culture infective dose (tcid ). results were expressed as tcid /ml and represent replicates obtained in four independent experiments (n = ). prior to molecular testing, viral dna was recovered from specimens using either homogenization with heat treatment as previously described [ ] , or using a commercial nucleic acid extraction as recommended by the manufacturer. for homogenization, μl of specimen and . g of various sized acid-washed silica beads: ≤ μm; - μm; - μm at a ratio of : : (sigma-aldrich, oakville, on) were placed on a fastprep- homogenizer (mp biomedicals, solon, oh) at . m/s for s. following a brief centrifugation at , × g for min, μl of the supernatant was diluted in two volumes of te buffer ( mm tris-hcl, mm edta, ph . ). the homogenate was then heated at °c for min, cooled to room temperature, and μl was subjected to adenovirus real-time pcr. automated extractions were performed on μl of specimen using a magna pure total nucleic acid isolation kit (roche diagnostics, mannheim, germany) on a roche magnapure lc instrument. the elution volume was μl. specimens with discordant results during method comparison were subjected to a manual dna extraction using a qiaamp dna blood mini kit (qiagen, toronto, on) with a sample volume of μl. the dna was eluted in μl, and concentrated -fold using a qiagen minelute pcr purification kit. plasmid dna, used for the internal control, was purified from a ml overnight culture using a qiaprep spin miniprep kit (qiagen) as recommended by the manufacturer. for molecular typing, amplicon was purified using a qiaquick gel extraction kit (qiagen) with a final elution volume of μl. all nucleic acid extractions were performed using manufacturers' instructions. nucleic acids were used immediately following extraction and aliquots were placed at − °c for longterm storage. the real-time pcr has been extensively validated using respiratory specimens [ ] . to facilitate workflow in the cdha microbiology laboratory (halifax, nova scotia, canada), the in-house assay was optimized for amplification and detection on a roche lightcycler . platform. real-time pcr was performed as duplex reactions with primers and probes (table ) targeting the adenovirus hexon gene and an exogenous internal control. for adenovirus, two sets of primers and probes were used to span the genetically diverse adenovirus types [ , ] . primers were synthesized by sigma genosys (oakville, on). probes for adenovirus and the internal control were purchased from biosearch technologies (novato, ca) and tib molbiol llc (adelphia, nj), respectively. the internal control, termed pgfp, is added to each reaction to monitor for the presence of pcr inhibitors. pgfp is a pmk-derived plasmid with a fragment of the gene encoding green fluorescence protein (gfp). the construct was synthesized, assembled, and transformed into escherichia coli k by life technologies (burlington, on). the final construct was verified by dna sequencing and restriction endonuclease digestion. e. coli harboring pgfp was inoculated into luria bertani broth supplemented with μg/ml kanamycin. plasmid dna was purified from a ml overnight culture and plasmid dna was quantified by spectrophotometry. ten-fold serial dilutions were used as template for the in-house realtime pcr. an inverse linear relationship (y = − . × + . ; r = . ) was generated by plotting crossing points (cp) values against plasmid concentration (data not shown). the linear range spanned cp values ranging from to , corresponding to concentrations of to copies per μl, respectively. for each pcr reaction, approximately copies were added. real-time pcr assay was performed using the light-cycler dna master hybprobe kit (roche diagnostics) in μl reactions consisting of: μl of template, × lightcycler faststart mix, mm mgcl ; . units of heat-labile uracil-n-glycosylase [ ] ; μl the internal control at copies/μl; nm of each adenovirus primer (adv f, adv r, adv f, adv r) and nm of probe (adv pr and adv pr); and nm of each pgfp primer (fgfp and rgfp) and nm of each probe (gfppr and gfppr ) ( table ). amplification and detection were performed using the lightcyler . instrument under the thermocycling conditions described for the roche hsv- / detection kit: initial activation at °c for min, followed by amplification cycles of denaturation at °c for s, annealing at °c for s, and elongation at °c for s. following amplification, melting temperature (tm) analysis was performed by measuring the fluorescent signal during the following cycling profile: °c for s, °c for s, and °c for s with a . °c/s transition. fluorescence was acquired at the annealing stage during amplification and continuously during the melting curve. cp and tm values were determined using software provided by the manufacturer. the nm (adenovirus) and nm (pgfp) channels were analyzed for presence or absence of target. pcr inhibition was suspected by either loss of positivity in the nm channel, or a shift in cp values greater than two standard deviations (cp ≥ . ) from the value obtained with the negative control. to resolve discrepant results obtained between the inhouse pcr assay and virus culture, or quantify the adenovirus dna during evaluation of the analytical sensitivity, the adenovirus r-gene kit (argene inc., sherley, ny) was used according to the manufacturer's protocol following a manual dna extraction. this internally controlled quantitative real-time pcr assay targets the hexon gene of adenovirus, and is validated for detection table nucleotide sequences of primers and probes used in this study sequence ( ′ to ′) reference cca gga cgc ctc gga gta [ ] adv r aaa ctt gtt att cag gct gaa gta cgt [ ] adv pr fam-agt ttg ccc gcg cca cca ccg -bhq * [ ] adv f gga cag gac gct tcg gag ta [ ] adv r ctt gtt ccc cag act gaa gta ggt [ ] adv pr fam-cag ttc gcc cgy gcm aca g -bhq * [ of types to [ ] . the kit contains: a ready-to-use premix contains (primers, probe, polymerase, and buffer) needed for amplification, quantification standards (at , , , , and , copies/reaction), and a sensitivity-control at copies/reaction. results were expressed as the number of copies per reaction. analytical specificity, limit of detection, and reproducibility the analytical specificity was first determined in silico by performing a basic local alignment search tool (blast) for primers, probes, and entire amplicon sequences using the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov). in addition, high titer nucleic acids were extracted from a panel of microorganisms chosen based on their ability to cause similar diseases or their potential for being found in the clinical specimen as a pathogen or normal flora ( (figure and table ). the analytical sensitivity (or limit of detection, lod) of the homogenization with heat treatment or nucleic acid extraction, in combination with the real-time pcr, was determined using -fold serial dilutions (in utm) of a cultured hadv-c type . each dilution was simultaneously processed by both extraction methods, and an aliquot immediately inoculated onto a cells for virus culture. the lod was defined by probit analysis [ ] using triplicate values obtained in four independent experiments by two different operators (n = ). each virus dilution was expressed as tcid /ml in the original sample. the virus dilutions were also quantified using a commercial real-time pcr and expressed as target copies/ reaction for each assay. intra-and inter-assay reproducibility were calculated for each dilution and expressed as % coefficients of variation (%cv). the performance of each method was compared to a modified gold standard to determine sensitivity, specificity, accuracy and precision. a case was defined by concordant results (positive or negative) between at least two assays. to resolve discrepant results obtained between the in-house real-time pcr assay and virus culture, dna was extracted manually and was subjected to commercial real-time pcr. the virus culture-positive specimens were subjected to pcr targeting the conserved segments surrounding the hypervariable region (hvr ) of the hexon gene terminator chemistry on the applied biosystems × l dna sequencer. type designation was undertaken by blast analysis, and confirmed by comparison to a database generated from sequences obtained from genbank [ ] . sequence analysis and multiple sequence alignments (clustalw analysis) were performed using the seqman and megalign components of lasergene software (dnastar, madison, wi). the phylogenetic tree was inferred using a neighbor-joining (nj) method with bootstrapping analysis for n = . chi-square and two-tailed fisher's exact tests were used to compare proportions in -by- contingency tables. confidence intervals ( %) for the estimated parameters are computed by a general method based on "constant chisquare boundaries" [ ] . agreement between assays was measured using kappa statistics. the statistical package for social sciences (spss) software v. was used and p ≤ . was used to denote a statistically significance. clades are shaded to depict species a to f. analytical specificity, limit of detection, and reproducibility blast searches of primers and probes targeting the adenovirus hexon gene the internal control sequences revealed that these were highly specific targets. in fact, no cross reactions were observed with high-titer nucleic acids extracted from other respiratory viruses or bacteria ( table ). the in-house real-time assay was able to detect serogroups a to f, including a variety of genetically diverse types: , , , , , , , , , , and ( figure and table ). as seen in figure , the performance of the inhouse pcr following the homogenization-or nucleic acid extraction-based protocols was equivalent. for each method, overlapping linear relationships were observed (y = − . × + . ; r = . compared to y = − . + . ; r = . , respectively) that spanned eight orders of magnitude with cp values ranging from to (figure a ). the intra-and inter-assay reproducibility of the real-time pcr following homogenization and heat treatment ranged from . to . %, and . to . %, respectively. similarly, intra-and inter-assay reproducibility of following the nucleic acid extraction protocol ranged from . to . % and . to . %. as expected, the highest %cv values observed for both methods were with virus dilutions near the lod. for hadv-c type , the lod for virus culture was . tcid /ml. the in-house real-time pcr was reproducibly positive following nucleic acid extraction or homogenization with viral stock dilutions corresponding to . tcid /ml ( / and / , respectively), and positive pcr reactions were frequently observed using virus dilutions of . tcid /ml ( / and / , respectively). virus stock dilutions were quantified using commercial real-time pcr assay, and the lod for homogenization or nucleic acid extraction-based protocols were shown to be approximately equivalent (figure ) . with a probability of %, the lod for the homogenization-and nucleic acid extraction-based protocols were copies/reaction (log = . ) and copies/reaction (log = . ), respectively ( figure b) . dilutions corresponding to the lod for virus culture were also quantified by real-time pcr and estimated at approximately copies/reaction ( figure b ). of the clinical specimens, concordant negative and concordant positive results were obtained when comparing virus culture to the in-house pcr following either of the two extraction methods ( figure a and table ). real-time pcr generated additional positive results that were later resolved as true positives using a manual dna extraction and a commercial real-time pcr ( figure a ). all pcr-positive culture-negative results were detected following homogenization protocol, whereas were detected following nucleic acid extraction ( figure a) . the single discordant result between the molecular assays had a cp value of . , suggesting that it may be attributed to sampling error (poisson distribution) at low concentrations of template [ ] . since the internal control also failed to amplify in this sample, the negative result could also be attributed to pcr inhibition. upon repeat processing by automated and manual nucleic acid extractions, positive results were obtained. therefore, the original specimen result was considered a false negative. overall, compared to the modified gold standard, the sensitivity of the inhouse real-time pcr following homogenization with heat treatment or nucleic acid extraction was approximately equivalent at % ( . - %) and . % ( . - . %), respectively (table ). in contrast, the sensitivity of virus culture was only . % ( . - . %) ( table ). the accuracy of each method was % ( . - %), . % ( . - . %), and . % ( . - . %), respectively ( table ) . all assays showed a high degree of specificity and precision (table ) . when comparing cp values for the positive results obtained with the real-time pcr following both extraction methods, a linear relationship was observed (y = . × + . ; r = . ) ( figure b ). cp values for homogenization with heat treatment were consistently higher than those obtained using the nucleic acid extraction; however, no significant differences in sensitivity (analytical or clinical) were observed (figure and table ). as expected, virus culture-positive specimens had positive pcr results with low cp values, whereas the virus culture-negative specimens had pcr-positive results with cp values greater than ( figure b ). dna extracted from the real-time pcr positive specimens were subjected to a conventional pcr targeting the conserved segments surrounding the hvr of the hexon gene [ ] . successful sequences were obtained from dna extracted from the specimens that were both virus culture and real-time pcr-positive. a type could be assigned using multiple sequence alignment of sequences derived from genbank, as previously described [ ] . individual blast analysis yielded similar results. three serogroups were observed: b (types , , , and ), c (types and ), and d (types , , and ). the predominant types observed were: ( . %), ( . %), ( . %), and ( . %). the conventional pcr was unable to amplify the target sequences from dna extracted from the virus culture-negative/real-time pcr-positive specimens. the cp values for these specimens ranged from to , suggesting only low quantities of virus were present (figure ). dna sequencing was also used to distinguish the prototypic hadv type p (strain de wit) from newly emerged type p . adenovirus type p has been associated with severe disease in europe and the north america [ ] [ ] [ ] [ ] . while the hexon hvr sequences obtained in this study share % identity with hadv type p , only two mutations (g a and g a) separate types p from p in this region. to further characterize the a b figure analytical sensitivity of the in-house real-time pcr. prior to amplification, -fold serial dilutions of hadv-c type were processed by homogenization and heat treatment (open circles, solid line), or nucleic acid extraction (filled squares, dashed line). in both cases, equivalent results were obtained in respect to: a) the linear range; and b) the lod determined by probit analysis (n = ). at a probability of %, the lod for the homogenization-and nucleic acid extraction-based protocols were copies/reaction (log = . ) and copies/reaction (log = . ), respectively. the same dilutions used for inoculate virus culture and dfa staining (indicated by open triangles, dotted line) were also quantified and demonstrated a lod of approximately copies/ml (log = . ). virus, the fibre knob gene was sequenced with primer pair f mut and r mut (table ) , using reaction conditions, thermocycling parameters, and dna sequencing as described for the molecular typing. compared to wild-type p, the fiber knob gene of hadv type p displays a -bp deletion (referred to as the k -e deletion) [ , , ] . the adenovirus type from this study harbored the characteristic -bp deletion, consistent with hadv type p (figure ) . an exogenous internal control was used in this study which is non-competitive (contains a primer pair that does not target adenovirus). the addition of the internal control and primers and probes to the in-house pcr reaction did affect the analytical sensitivity of the assay (data not shown). since the internal control was added at the level of pcr, both extraction methods could be directly evaluated for the presence of pcr inhibitors. despite a subsequent heat treatment and dilution step, homogenization is a crude method to recover viral dna and may not sufficient to remove or inactivate pcr inhibitors. amplification of the internal control in adenovirusnegative specimens is consistent with a true negative result and not simply attributed to pcr inhibition. pcr inhibition was suspected by either loss of positivity in the nm channel, or a shift in cp values greater than two standard deviations (which corresponds to approximately ± . cp) from the value obtained with the negative control. this value was established previously, where the internal control cp values from consecutive hsv-negative specimens were compared by homogenization and heat treatment or nucleic acid extraction [ ] . this cutoff value remains true for the internal control used in this study. since the in-house pcr was performed as a duplex with an internal control added at the level of pcr, the clinical specimens processed following homogenization and heat treatment or nucleic acid extraction could be monitored directly for the presence of potential pcr inhibitors. potential inhibitory substances were observed in two distinct cases: the first was a specimen that had been processed by homogenization with heat treatment, and the second, in a specimen subjected to nucleic acid extraction. in both cases, pcr inhibition was not observed upon repeat processing, suggesting either a processing error had occurred or the pcr inhibitor was labile [ ] . therefore, pcr inhibition could not be proven or excluded. as a result, the rate of possible pcr inhibition with either extraction method was equivalent at . % ( / ). at cdha (halifax, ns, canada), the average number of specimens submitted yearly for adenovirus testing is (range to for years to ) and the turnaround time for virus culture can be up to days. a cost analysis was performed that assumed a more practical approach of bi-weekly molecular testing ( - specimens with positive, negative and reagent controls). excluding labor, the average cost of a commercial pcr following nucleic acid extraction would range from $ to $ (cad) per specimen. in comparison, the inhouse real-time pcr following a nucleic acid extraction would reduce the cost approximately~ -fold ($ . to $ . ). replacement of the nucleic acid extraction with the homogenization-base protocol further reduces the cost~ -fold ($ . to $ . ), which is comparable to the average cost of virus culture ($ . to . ). the time require for bi-weekly processing for either molecular methods is~ h/week, which is far lower than the time required for weekly maintenance and processing of specimens using cell culture and dfa staining. naats like real-time pcr have revolutionized the detection of human pathogens in clinical microbiology laboratories. rapid specimen throughput and excellent performance characteristics make them an appealing alternative to traditional culture methods; however, cost limits their use in many clinical laboratories. both the recovery of nucleic acids using extraction and the pcr reaction itself contribute to the cost. we have shown that combining a crude extraction method like homogenization with heat treatment [ ] and an in-house real-time pcr [ ] is a cost effective strategy for the detection hadv from swabs submitted in utm. homogenization uses multidirectional motion to disrupt cells through contact with silica beads and the heat treatment [ , ] . in combination with a subsequent heat treatment to inactivate heat-labile pcr inhibitors, this crude mechanical lysis had been shown to be a cost-effective method to recover viral dna from swabs transported in utm [ ] . the performance characteristics of this approach were equivalent to using traditional nucleic acid extraction and both molecular methods far exceeded those obtained with virus culture. replacing the nucleic acid extraction with the homogenization protocol did not affect the analytical (or clinical) sensitivity of the real-time pcr (figure and table ). using dilutions of hadv-c type , the lod for the homogenization protocol was approximately copies/reaction, was consistent with previously reported values ( - copies/reaction) for hadv types and [ ] . this analytical sensitivity is approximately -fold more sensitive than the estimated lod for virus culture. furthermore, positive results could be even obtained at copies/reaction with a probability of . % ( figure b ). while no significant differences were observed between the molecular assays, both demonstrated a high level of analytical sensitivity. when comparing clinical specimens using a modified gold standard, the in-house pcr following homogenization and heat treatment or nucleic acid extraction demonstrated similar sensitivities of % and . %, respectively (table ). this far surpasses the performance of virus culture at . %. the % increase in positivity is consistent with the~ -fold increase in analytical sensitivity and is not surprising since similar results were observed when transitioning other viruses from culture to naats [ ] [ ] [ ] [ ] . when comparing positive results from the in-house real-time pcr, cp values obtained following the homogenization protocol were consistently higher than those obtained following nucleic acid extraction ( figure b ). however, the analytical and clinical sensitivities of each assay were not significantly different ( figure and table ). it should be noted that all virus culture-negative/pcr-positive specimens had cp values greater than , corresponding to viral loads that fell below the lod for virus culture ( figure b ). the homogenization-or nucleic acid extraction-based protocols both showed excellent analytical specificity, with no cross-reactions from other organisms ( table ) . both methods were able to detect diverse hadv types spanning all the different species (figure and table ). of the virus culture-positive specimens, the most predominant types detected were , and , belonging to species b, d and c, respectively. these hadv types are well-recognized causes of acute respiratory tract and ocular infections and are consistent with the distribution reported by others regions in canada [ , ] . interestingly, a variant of hadv type , termed p , has been described as an emerging pathogen associated with outbreaks and sporadic cases of acute respiratory disease in europe and the united states [ ] [ ] [ ] [ ] . while most recorded cases were mild infections, severe disease and deaths have occurred. hadv type p has a characteristic -bp deletion (k -e ) in the fiber knob gene [ , , ] . the adenovirus type from this study was consistent with type p and harbored these mutations ( figure ). while there has been a number of reports of type p circulating in the us and europe, this variant has only once been reported in canada [ ] . the first adenovirus p cases in canada were reported from nova scotia's neighboring province, new brunswick, and included one fatality (figure ) [ ] . the specimen identified as p in this study was obtained from a fatal case dating back to same time period as the new brunswick cases. further epidemiological investigations are underway. while severe and fatal cases associated with type p have been reported, similar outcomes have been reported with many other common hadv types [ , , , ] . the most likely culprit of disease severity is the immune status of the host, not the adenovirus type or species. it should be noted that the thermocycling conditions for the adenovirus pcr were modified to allow simultaneous processing of other real-time pcr assays (hsv and vzv) in the cdha microbiology laboratory [ ] . simultaneously processing of multiple pcr assays on the same lightcycler instrument allows more efficient batch testing when equipment availability is limited. interestingly, these modifications allowed the detection of hadv type which had previously been problematic on an abi instrument [ ] . difference between assays can be attributed to a numerous factors (i.e. instrumentation, kits, etc.); however, the most likely explanation in this case is the annealing temperature. using the original pcr protocol [ ] , hadv type could only be detected when the annealing temperature was reduced from °c to °c [ ] . the annealing temperature in this study is °c. using conditions described in this study, the detection of hadv type has now been replicated in both collaborating laboratories. a limitation of this study is that the validation of homogenization was only performed using swabs in utm. future experiments will need to examine whether homogenization can be applied to other relevant specimen types (urine, stool, blood and tissue); however, the realtime pcr following a nucleic acid extraction has been shown to be effective for this purpose [ , ] . secondly, the performance characteristics of homogenization may vary between pcr assays and should not be implemented without proper validation [ ] . while homogenization with heat treatment has shown to be effective for the recovery of viral dna from hadv (this study), hsv [ ] , and varicella zoster virus, decreased sensitivity was observed for enveloped rna viruses like mumps and influenza viruses ( [ , ] leblanc, j. unpublished data). homogenization 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serves both clinical and fundamental virology real-time qualitative pcr for human adenovirus types from multiple specimen sources development of a pcrbased assay for detection, quantification, and genotyping of human adenoviruses molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time pcr assay multiplexed, realtime pcr for quantitative detection of human adenovirus pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr evaluation of type-specific real-time pcr assays using the lightcycler and j.b.a.i.d.s. for detection of adenoviruses in species hadv-c homogenization with heat treatment: a cost effective alternative to nucleic acid extraction for herpes simplex virus real-time pcr from viral swabs a reliable and inexpensive method of nucleic acid extraction for the pcr-based detection of diverse plant pathogens presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (a ) cell culture uracil-dna glycosylase (ung) influences the melting curve profiles of herpes simplex virus (hsv) hybridization probes probit analysis comprehensive detection and serotyping of human adenoviruses by pcr and sequencing logistic regression quantitation of targets for pcr by use of limiting dilution genome sequences of human adenovirus isolates from mild respiratory cases and a fatal pneumonia, isolated during - epidemics in north america genome sequence of the first human adenovirus type isolated in china inhibition and facilitation of nucleic acid amplification a comparison of cell culture versus real-time pcr for the detection of hsv / from routine clinical specimens adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis comparison of a commercial qualitative real-time rt-pcr kit with direct immunofluorescence assay (dfa) and cell culture for detection of influenza a and b in children efficacy of pcr and other diagnostic methods for the detection of respiratory adenoviral infections epidemiology of severe pediatric adenovirus lower respiratory tract infections in manitoba, canada characterization of culture-positive adenovirus serotypes from respiratory specimens in genome type analysis of adenovirus types and isolated during successive outbreaks of lower respiratory tract infections in children detection of mumps virus rna by real-time one-step reverse transcriptase pcr using the lightcycler platform viral agents of human disease: biosafety concerns a cost effective real-time pcr for the detection of adenovirus from viral swabs we would like to thank members of division of microbiology, department of pathology and laboratory medicine at cdha (halifax, nova scotia) for their ongoing support and for funding for this project. in particular, we are indebted to wanda brewer for the propagation and maintenance of a cells, and the various technologists responsible for routine virus culture. the authors declare that they have no competing interests.authors' contributions jl conceived the study. jl, th and rt participated in its design and coordination. ta, kb, and jl carried out the molecular testing. mw quantified the adenovirus stocks and established tcid values. ta and kb performed statistical analyses. jl analyzed the dna sequencing results. rt, sw and kp were involved in the phylogenetic analyses and typing of the adenoviruses as well as preparing the specificity panels. all authors were involved in the preparation of the manuscript. all authors have read and approved the final manuscript. key: cord- -ajlctjeb authors: golden, joseph w; schiff, leslie a title: neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in u promonocyte cells date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ajlctjeb background: mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. during enteric infections, proteolysis of the reovirus outer capsid protein σ is mediated by pancreatic serine proteases. in contrast, the proteases critical for reovirus replication in the lung are unknown. neutrophil elastase (ne) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. in addition to its normal role in microbial defense, aberrant expression of ne has been implicated in the pathology of acute respiratory distress syndrome (ards). because reovirus replication in rodent lungs causes ards-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of ne to promote reovirus virion uncoating. results: the human promonocyte cell line u expresses ne. treatment of u cells with the broad-spectrum cysteine-protease inhibitor e [trans-epoxysuccinyl-l-leucylamido-( -guanidino)butane] and with agents that increase vesicular ph did not inhibit reovirus replication. even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in u cell cultures. to identify the protease(s) responsible, u cells were treated with phorbol -myristate -acetate (pma), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including ne and cathepsin (cat) g. in the presence of e , reovirus did not replicate efficiently in pma-treated cells. to directly assess the role of ne in reovirus infection of u cells, we examined viral growth in the presence of n-ala-ala-pro-val chloromethylketone, a ne-specific inhibitor. reovirus replication in the presence of e was significantly reduced by treatment of cells with the ne inhibitor. incubation of virions with purified ne resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis. conclusion: our findings reveal that ne can facilitate reovirus infection. the fact that it does so in the presence of agents that raise vesicular ph supports a model in which the requirement for acidic ph during infection reflects the conditions required for optimal protease activity. the capacity of reovirus to exploit ne may impact viral replication in the lung and other tissues during natural infections. mammalian reoviruses are the prototypic members of the reoviridae family, which also includes the pathogenic rotaviruses, coltiviruses, seadornaviruses and orbiviruses. these viruses share elements of their replication cycle as well as structural features, including a non-enveloped multi-layered capsid that surrounds a segmented dsrna genome. in humans, mammalian reoviruses are typically associated with mild and self-limiting enteric and respiratory infections. however, studies in neonatal mice reveal that reoviruses can spread to distant tissue sites in immunocompromised hosts (reviewed in [ ] ). the factors that determine reovirus cellular host range are poorly understood. because reovirus attaches to cells through interactions with broadly expressed receptors, one or more subsequent steps in the viral life cycle must help to regulate host range and pathogenesis. our recent studies suggest that one such step is proteolysis of the capsid protein σ [ , ] . in cell culture, the first step in infection is attachment to cellular receptors through interactions with the viral protein σ [ , ] . σ interacts with two known receptors: sialic acid and junctional adhesion molecule [ ] [ ] [ ] . following binding, virions are internalized by receptor-mediated endocytosis [ ] . endocytosis is an essential step in the viral life cycle under standard infection conditions [ ] . within the endosomal and/or lysosomal compartment, proteases convert virions into particles that resemble in vitro-generated intermediate subvirion particles (isvps) [ ] [ ] [ ] [ ] [ ] . these uncoating intermediates, typically prepared using chymotrypsin or trypsin, lack σ and have a cleaved form of µ . studies using isvps and isvps recoated with recombinant outer capsid proteins reveal that σ plays a key role in regulating reovirus cell entry by interacting with, protecting, and controlling the conformational status of the underlying penetration protein µ [ ] [ ] [ ] [ ] . in cells that cannot efficiently mediate σ degradation during uncoating, reovirus infection is slow or blocked; these cells can be productively infected by particles that lack σ [ ] . in vitro, isvp-like particles can be generated by a variety of proteases in addition to chymotrypsin and trypsin, including proteinase k, thermolysin, endoproteinase lys-c, cat l, cat b and cat s [ , [ ] [ ] [ ] . recent work has provided insight into the cellular determinants of reovirus uncoating. in murine fibroblasts, where reovirus entry has been best studied, the cysteine proteases cat l, and to a lesser extent cat b, are required for σ removal, whereas the aspartyl protease cat d is not [ , [ ] [ ] [ ] [ ] [ ] . virion disassembly in murine fibroblasts also requires acidic ph [ , , ] . recently, we demonstrated that reovirus uncoating in the macrophage-like cell line p d is mediated by the acid-independent lysosomal cysteine protease cat s [ ] . this finding revealed that in different cell types, distinct proteases can facilitate reovirus uncoating. our results suggested a model in which infection in some cells is acid-dependent because the proteases that mediate σ removal in those cells require acidic ph for maximal activity. thus, in fibroblasts or other cells in which the acid-dependent proteases cat l and cat b mediate σ removal, infection is acid-dependent [ , , ] , whereas in cat s-expressing cells it is not [ ] , because cat s maintains its activity at neutral ph [ ] . insight from the analysis of reovirus cell entry facilitated the recent discovery that activation of the ebola virus glycoprotein also depends on the activity of the acid-dependent endosomal proteases cat b and cat l [ ] . the role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread, and disease in animals is largely unknown, except in the murine intestinal tract where pancreatic serine proteases have been shown to mediate σ removal [ , ] . reovirus also naturally infects hosts via the respiratory tract [ ] [ ] [ ] . one protease with well-described effects in the respiratory tract is elastase (genbank nm_ ), an inflammatory serine protease of the chymotrypsin family, which is predominantly expressed by neutrophils [ ] . ne plays a prominent role in wound repair [ ] [ ] [ ] and in controlling microbial infections [ ] [ ] [ ] . ne expression can also promote pathogenesis; it has been implicated in smokeinduced emphysema [ ] , respiratory syncytial viral bronchiolitis [ ] and in the respiratory syndrome ards [ ] . the fact that reovirus replication in the rodent lung causes an influx of neutrophils [ , ] and that reovirus infection can recapitulate ards [ ] , led us to ask whether ne could mediate productive reovirus uncoating. we investigated reovirus infection in the monocyte-like cell line u , because it is known to express ne [ ] . experiments described in this report demonstrate that reovirus infection in u cells does not require cysteine protease activity and is not blocked in the presence of agents that raise vesicular ph. studies using protease inhibitors suggest that, in the absence of cysteine protease activity, ne is largely responsible for productive infection of u cells. ne can directly mediate σ removal from reovirus virions; the resultant particles are infectious and do not require additional intracellular proteolysis. our data raise the possibility that ne is involved in reovirus replication in the respiratory tract. analysis of viral replication in l and u cells treated with e figure analysis of viral replication in l and u cells treated with e . a. × l and u cells were untreated (-; black) or treated (+; grey) with µm e for h or d. cysteine protease activity was assessed using the fluorogenic substrate z-phe-arg-mca (sigma) and plotted in arbitrary units. activity levels in treated cells were so low (in l cells, units at h and units at d; in u cells, units at h and units at days) that they cannot be visualized on this graph. b. l (l; black bars) and u (u; grey bars) cells were treated with µm e for h prior to infection. cells were then infected with reovirus strain lang virions or isvps at an moi of . infectious virus present at d p.i. was determined by plaque assay on l cell monolayers. each time point represents the mean (+/-sd) derived from three independent samples. virion isvp we first established conditions under which lysosomal cysteine protease activity was inhibited. cells were treated with µm e , a broad-spectrum cysteine protease inhibitor [ ] , and protease activity was assessed using the cat l and cat b-specific fluorogenic substrate z-phe-arg-mca. we analyzed enzyme activity at two time points: first after h of treatment, because we typically pre-treat cells with inhibitors for h prior to infection, and second at d, the time point at which viral yield would be quantified. as shown in fig. a , treatment with µm e completely abolished cysteine protease activity in u cells. consistent with our previous findings [ ] , e also completely blocked cysteine protease activity in l cells. raw values are provided, to illustrate the relative difference in cat l/b enzyme activity levels between u cells and l fibroblasts. in the absence of inhibitor, cat l and b activity was significantly lower in u cells than in l cells. this may be a consequence of high expression in u cells of cystatin f, an intracellular cysteine protease inhibitor with specificity for cat l and papain [ ] . next, we compared reovirus replication in e -treated u and l cells. cells were pre-treated for h and infected with lang virions or isvps at a multiplicity of infection (moi) of . the results of a representative experiment are shown in fig. b . in the absence of e , both l and u cells supported reovirus replication, consistent with the fact that these cells express cat l. as expected, e blocked virion infection of l cells; however, viral yields in e -treated u cells were only slightly reduced relative to untreated cells. isvps, which lack capsid protein σ , replicated efficiently in treated cells, indicating that µm e was not toxic to either cell type. these results demonstrate that productive infection of u cells by lang virions does not require the activity of e -sensitive, papain-like cysteine proteases. acidic ph is required for productive reovirus infection of murine l fibroblasts [ , ] , in which the aciddependent proteases cat l and cat b mediate uncoating [ , ] . serine proteases, including ne, and metalloproteases function over a broader ph range. therefore, to gain insight into the nature of the protease(s) that can promote reovirus uncoating in u cells, we investigated the requirements for acidic ph. l and u cells were left untreated or pre-treated with e in the presence or absence of bafilomycin a (baf) or nh cl. these latter agents raise vesicular ph by blocking the vacuolar h + -atpase pump or by acting as a weak base, respectively [ ] [ ] [ ] . after pre-treatment, cells were infected with lang virions at an moi of and viral yields were determined at days post infection (d p.i.). a representative experiment is shown in fig. . treatment with either baf or nh cl did not inhibit viral replication in u cells; yields reached . and . logs, respectively. furthermore, these agents had little effect on viral replication in u cells even when the cells were also treated with e to inhibit cysteine protease activity. in contrast, baf or nh cl alone completely blocked reovirus replication in l cells, consistent with the requirement for cat l/b-mediated σ removal in these cells. given that reovirus uncoating is an essential step in the viral life cycle [ ] , these findings revealed that a non-cysteine protease that functions at neutral ph can facilitate this step in u cells. treatment of the promonocytic u cells with phorbol ester derivatives results in their differentiation into macrophage-like cells [ , ] . this differentiation is characterized by several major phenotypic changes, including increases in expression of urokinase plasminogen activator receptors, upregulation of collagenase activity and a significant decrease in the expression of ne and cat g [ , ] . we predicted, therefore, that pma treatment might decrease the capacity of reovirus virions to replicate in u cells when cysteine proteases were inhibited. to confirm that there was a significant decrease in ne expression in u cells differentiated with pma, u cells were treated with nm pma for h and expression of ne was analyzed by immunoblotting. as shown in fig. a , ne was expressed in untreated u cells, but its expression was dramatically reduced following pmainduced differentiation. to examine the effect of u cell differentiation on reovirus infection, pma-treated and untreated u cells were left untreated or were treated with e for h and infected with lang virions or isvps at an moi of . yields were measured at d p.i. and the results of a typical experiment are shown in fig. b . in the absence of e , pmatreated u cells were permissive to infection by virions. pma treatment only decreased yields by ~ . log relative to untreated cells. in contrast, when pma-differentiated u cells were treated with e to inhibit cysteine protease activity, they no longer supported productive infection by lang virions. because these results could be explained if e was toxic to pma-treated u cells, we examined the replication of isvps. in the presence of e , isvps replicated to high yields in both undifferentiated and differentiated u cells. since pma-induced differentiation of u cells caused a substantial decrease in ne expression, these results are consistent with the hypothesis that ne or another similarly regulated neutral protease facilitates productive reovirus infection in promonocytic (pre-differentiated) u cells. effects of agents that raise vesicular ph on reovirus replication in u and l cells a. analysis of reovirus replication in u cells differentiated with pma figure analysis of reovirus replication in u cells differentiated with pma. a. lysates generated from u cells that were untreated (-) or treated with nm pma for h were resolved on sds- % polyacrylamide gels and electrophoretically transferred to a nitrocellulose filter. the filter was subsequently incubated with a polyclonal goat antibody against human ne ( : ) (santa cruz biotechnology). the filter was washed and incubated with a secondary anti-goat antibody conjugated to horseradish peroxidase ( : ) (santa cruz biotechnology). protein bands were detected using reagents that generate a chemiluminescent signal (amersham). b. u cells that were undifferentiated (-; black bars) or differentiated (pma; grey bars) with nm of pma for h were left untreated (-) or were treated with µm e . following pre-treatment with the protease inhibitor, cells were infected with lang virions or isvps at an moi of . viral yield was quantified at d p.i. as described in the legend to fig. b. a. b. we directly examined the capacity of ne to facilitate reovirus infection by using the irreversible elastase inhibitor, n-(methoxysuccinyl)-ala-ala-pro-val-chloromethyl ketone [ ] . this inhibitor is highly specific for ne and does not inhibit the activity of the related serine protease, cat g [ ] . first, we established the efficacy and specificity of inhibitor treatment under our experimental conditions. u cells were treated with the ne inhibitor, e , baf or nh cl for either h or d and the activity of ne in cell lysates was examined using a colorimetric substrate. as shown in table , the ne inhibitor was active at both time points. in cells treated with the specific inhibitor, ne activity was less than % of that in untreated u cells. in contrast, in u cells treated with e , baf or nh cl, ne activity was only modestly reduced, remaining above % even after d. these results are consistent with the capacity of ne to function at neutral ph. to verify the specificity of the ne inhibitor, we also examined its effect on cat l/b activity using the fluorogenic substrate z-phe-arg-mca. as expected, cat l/b activity was completely inhibited by e but largely unaffected by the ne inhibitor. to examine the effect of the ne inhibitor on reovirus replication in u cells, we pre-treated them for h with e in the presence or absence of the ne inhibitor, infected them with lang virions or isvps at an moi of , and quantified viral yields at d p.i. a representative experiment is shown in fig. . consistent with the results shown in fig , virion replication was not blocked in e treated u cells. however, in the presence of both e and the ne inhibitor, yields were significantly reduced. isvps replicated to high yields in treated cells, indicating that the combination of inhibitors was not toxic to u cells. these results demonstrate that ne plays a critical role in reovirus infection of u cells when cysteine proteases are inhibited. ne, like many cellular proteases, is expressed as a proenzyme that becomes activated only after its pro-region is removed [ ] . we envisioned two models by which ne could facilitate reovirus infection of u cells. in the first, ne could directly mediate σ degradation, leading to the generation of an isvp-like particle. in the second, ne could act indirectly by activating another protease. to try to distinguish between these models, we examined the capacity of purified ne to directly mediate σ removal from lang virions in vitro. purified lang virions were treated with ne for and h and the treated virus particles were analyzed by sds-page. as shown in fig. a , ne efficiently removed σ from lang virions; after h very little intact σ remained on viral particles. after h of ne treatment, σ was completely removed and the underlying µ c was cleaved to the δ and φ fragments (φ was not retained on the gel). when we assayed the infectivity of the resultant particles by plaque assay we found that ne treatment did not negatively affect the titer of lang particles (data not shown). to determine if ne-generated svps required further proteolytic processing of σ , l cells were pre-treated with e to block cysteine protease activity and infected at an moi of with lang virions, isvps or ne-generated subviral particles (ne-svps). viral yields were determined at d p.i. as expected, e blocked infection of l cells by virions. in contrast, both isvps and ne-svps replicated efficiently in the presence of the cysteine protease inhibitor (fig. b) . because virion disassembly in l cells requires acidic ph [ ] , we also examined the capacity of ne-svps to infect l cells treated with baf, nh cl or monensin, three agents that raise vesicular ph by distinct mechanisms. cells were treated with these agents and then infected with virions, isvps or ne-svps at an moi of a u cells were treated with the indicated inhibitors for h or d. b ne activity was assessed using the colorimetric substrate meosuc-ala-ala-pro-val-ρna and percent activity relative to untreated cells was calculated. c cathepsin l and b activity were assessed using the fluorogenic substrate z-phe-arg-mca and percent activity relative to untreated cells was calculated. . at hours post infection (h p.i.), cell lysates were harvested and expression of the reovirus non-structural protein µns was analyzed by immunoblotting (fig. c ). as expected, when treated cells were infected with virions, viral protein expression was blocked. in contrast, µns expression was evident even in the presence of agents that raise ph when infections were initiated with isvps or ne-svps (fig. c ). together, these results demonstrate that ne can directly mediate σ removal from virions to generate infectious particles that do not require further proteolytic processing by acid-dependent cysteine proteases in l cells. serine proteases are involved in reovirus infection in the mammalian intestinal tract [ ] and in this report we pro-vide evidence that they can mediate uncoating and promote infection in u cells. this expands the range of proteases that promote reovirus infection in cell culture to include ne as well as the cysteine proteases cat l, cat b, and cat s. several lines of evidence now support the notion that protease expression is a cell-specific host factor that can impact reovirus infection. for example, some reovirus strains are inefficiently uncoated by cat s and thus do not replicate to high yield in p d macrophages [ ] . in this report we demonstrate that pma-induced differentiation influences the type of protease that mediates reovirus uncoating in u cells. in these cells, pma treatment is reported to increase cat l expression [ ] and decrease expression of the serine proteases ne and cat g [ , ] . accordingly, when we used pma to induce u cell cultures to differentiate, reovirus infection became sensitive to the cysteine protease inhibitor e . we suspect that cat l is largely responsible for uncoating in these pma-differentiated cells, but the acid-independent protease cat s may also play a role. we are currently addressing this question by analyzing infection in pmadifferentiated cells treated with either baf or nh cl. our data do not completely resolve this question. cat g is expressed by u cells and, like ne, it is down-regulated by pma treatment. furthermore, we found that in vitro treatment of reovirus virions with purified cat g generates svps that behave like ne-svps in that they are infectious in the absence of further proteolytic processing (data not shown). results of our experiment with the ne-specific inhibitor suggest that ne is largely responsible for the e -resistant infection in u cells. while this inhibitor is reported not to inhibit cat g [ ] , we have not independently confirmed this. another approach to assess the role of cat g in reovirus infection of u cells would be to examine the effect of cat g-specific inhibitors on infection. we tried one such inhibitor, cathepsin g inhibitor i (calbiochem) [ ] , but found that it was cytotoxic to u cell cultures. given that both ne and cat g can generate infectious reovirus svps, more work needs to be done in order to understand the role that these two proteases play in infection in these cells. previously, we reported that virion uncoating mediated by cat s does not require acidic ph [ ] . these results were consistent with the acid-independence of cat s activity [ ] . together, the results in fig. and fig. reveal that, like cat s, ne-mediates infection in an acid-independent manner. this finding thus provides further support for a model in which the requirement for acidic ph during reovirus infection of some cell types reflects the requirement for acid-dependent protease activity in those cells rather than some other requisite acid-dependent aspect of cell entry. the small effect of baf and nh cl on e -resistant reovirus growth (fig. ) may reflect the participation of one or more acid-dependent proteases (such as cat d) in the activation of ne. elastase is stored in azurophilic granules that are the major source of acid-dependent hydrolases in neutrophils [ ] . although these granules do not contain lamp- or lamp- [ ] they contain the lysosomal markers lamp- [ ] and cd [ ] and are accessible to endocytosed fluid-phase markers under conditions of cellular stimulation [ ] . ne can be released from neutrophils during degranulation [ ] and its cell surface expression can be induced upon pma treatment [ ] . however, studies in u cells have shown that ne is predominantly retained intracellularly and that little if any activity is present in the extracellular medium [ ] . consistent with this, we have been unable to generate isvp-like particles by treatment of virions with u culture supernatants (data not shown). this observation, together with our finding that pma treatment decreases the capacity of e -treated u cells to support reovirus infection, leads us to favor a model in which ne-mediated virion uncoating in u cell cultures occurs intracellularly. in vivo, a number of viruses, including dengue and respiratory syncytial virus, induce the release of il- , a cytokine that serves as a chemoattractant for neutrophils and promotes their degranulation [ , ] . reovirus replication in the rat lung results in neutrophilic invasion [ , ] and studies in cell culture indicate that reovirus infection can induce il- expression [ ] . thus, the capacity of reovirus to induce il- secretion in vivo might facilitate the release of neutrophilic lysosomal hydrolases, including ne, into the extracellular milieu. in this report, we have shown that mammalian reovirus can utilize this acid-independent serine protease for uncoating. our data suggest that, in vivo, one consequence of reovirus-induced il- expression would be the generation of infectious ne-svps. like isvps, these particles would be predicted to have an expanded cellular host range because they can infect cells that restrict intracellular uncoating [ ] . thus, inflammation might be predicted to exacerbate reovirus infection by promoting viral spread. future studies using mice with deletions in the ne gene will be required to elucidate the role this protease plays during reovirus infection in the respiratory tract and other tissues. finally, given the recent finding that endosomal proteolysis of the ebola virus glycoprotein is necessary for infection [ ] , our results raise the interesting possibility that ne or other neutrophil proteases may play a role in cell entry of other viruses. [furlong, # ] . isvps were prepared by treating purified virions with chymotrypsin as described elsewhere [nibert, # ] . cysteine protease activity was measured as described previously [ ] samples were frozen and thawed three times and titrated by plaque assay on l cells as described elsewhere [ ] . viral yields were calculated according to the following formula: log (pfu/ml) t = x hrlog (pfu/ml) t = +/-standard deviation (sd). to analyze ne expression, cell lysates were generated from u cells, either treated or untreated for h with nm pma as described for the analysis of viral protein expression. lysate from the equivalent of × cells was run on sds- % polyacrylamide gels and transferred to nitrocellulose. membranes were blocked overnight in tbst containing % nonfat dry milk. ne expression was analyzed using a polyclonal antibody against ne ( : in tbst) (santa cruz biotechnology inc, santa cruz, ca). membranes were washed with tbst and incubated with a horseradish peroxidase-conjugated anti-goat igg ( : in tbst). bound antibody was detected by treating the nitrocellulose filters with enhanced chemiluminescence (ecl) detection reagents (amersham) and exposing them to full speed blue x-ray film (henry schein, melville, ny). cells were plated at /well in a -well plate - h prior to infection. virus was allowed to adsorb to cells for . h at °c. at this temperature, virus binds to cells but is not internalized [ ] . after adsorption, the cultures were incubated at °c in fresh medium. prior to some infections, cells were pre-treated for h with µm e , nm baf, µm monensin (sigma), or mm nh cl. in those instances inhibitors were also included in the post-adsorption culture medium. at the indicated times p.i., cells were collected by centrifugation at × g, washed twice in chilled pbs and lysed in tlb. after centrifugation at × g to remove cellular debris, samples were resuspended in sample buffer. protein samples (representing × cells) were analyzed by electrophoresis on sds- % polyacrylamide gels and transferred to nitrocellulose membranes for h at v in mm tris- mm glycine- % methanol. nitrocellulose membranes (bio-rad laboratories, hercules, calif.) were blocked overnight at °c in tbst ( mm tris [ph . ], mm nacl and . % tween) containing % nonfat dry milk, rinsed with tbst, and incubated with a rabbit anti-µns polyclonal antiserum [ ] ( : in tbst) for h. membranes were subsequently washed with tbst and incubated for h with horseradish peroxidase-conjugated anti-rabbit immunogloblin g (igg) ( : in tbst) (amersham, arlington heights, ill.). bound antibody was detected by treating the nitrocellulose filters with enhanced chemilumescence (ecl) detection reagents (amersham) and exposing the filters to full speed blue x-ray film (eastman kodak, rochester, n.y.). purified virions ( . × ) were incubated with µg/ ml of purified neutrophil elastase (calbiochem) in µl of vdb at °c for h. reactions were terminated by adding mm pmsf and µm ne inhibitor to the reaction mixture. . × particles were run on sds- % polyacrylamide gels stained with coomassie brilliant blue to confirm the removal of σ . viral infectivity was determined by plaque assay on l cell monlayers. purified lang virions ( . × ) were treated with µg/ ml of ne in µl of vdb at °c for the times indicated. reactions were terminated as described above. to verify σ removal, the proteins from . × particles were separated on sds- % polyacrylamide gels and visualized with coomassie brilliant blue staining. viral infectivity for each time point was determined by plaque assay on l cell monolayers. reoviruses. in fields virology addition of exogenous protease facilitates reovirus infection in many restrictive cells cathepsin s supports acid-independent infection by some reoviruses protein sigma is the reovirus cell attachment protein absolute linkage of virulence and central nervous system cell tropism of reoviruses to viral hemagglutinin utilization of sialic acid as a coreceptor enhances 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cells l-trans-epoxysuccinyl-leucylamido( -guanidino)butane (e- ) and its analogues as inhibitors of cysteine proteinases including cathepsins b, h and l regulated expression and intracellular localization of cystatin f in human u cells bafilomycins: a class of inhibitors of membrane atpases from microorganisms, animal cells, and plant cells weak bases and ionophores rapidly and reversibly raise the ph of endocytic vesicles in cultured mouse fibroblasts fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation regulation of urokinase receptors in monocytelike u cells by phorbol ester phorbol myristate acetate specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin g. inhibition with peptide chloromethyl ketones an unusual specificity in the activation of neutrophil serine proteinase zymogens phorbol ester stimulated cathepsin l expression in u cells developmental regulation of the human cathepsin g gene in myelomonocytic cells transcriptional and posttranscriptional modulation of human neutrophil elastase gene expression nonpeptide inhibitors of cathepsin g: optimization of a novel beta-ketophosphonic acid lead by structure-based drug design exploring cells with a centrifuge the lysosomal membrane glycoproteins lamp- and lamp- are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation ultrastructural localization of the cd macrophageassociated antigen in human blood neutrophils and monocytes linkage of azurophil granule secretion in neutrophils to chloride ion transport and endosomal transcytosis the neutrophil : cellular biochemistry and physiology cytokines regulate membrane-bound leukocyte elastase on neutrophils: a novel mechanism for effector activity inflammatory mediators in dengue virus infection in children: interleukin- and its relationship to neutrophil degranulation respiratory syncytial virus stimulates neutrophil degranulation and chemokine release prolonged induction of il- gene expression in a human fibroblast cell line infected with reovirus serotype strain lang sigma protein of mammalian reoviruses extends from the surfaces of viral particles the penetration of reovirus rna and initiation of its genetic function in l-strain fibroblasts reovirus nonstructural protein muns binds to core particles but does not inhibit their transcription and capping activities we express thanks to stephen rice and max nibert for critically reviewing this manuscript. we also thank members of the schiff, rice and bresnahan laboratories for their constructive input throughout these studies. this work was supported by nih grant ai and university of minnesota graduate school grant-in-aid # to l. a. s. j. w. g. received support from nih training grant t ai . the author(s) declare that they have no competing interests. j.w.g. performed all experiments and was responsible for the experimental design and data analysis. j.w.g. also wrote the initial draft of the manuscript. l.a.s. is corresponding author, participated in experimental design, data analysis and critically edited the manuscript. both authors read and approved the final manuscript. key: cord- -f prnpsu authors: eichler, robert; lenz, oliver; garten, wolfgang; strecker, thomas title: the role of single n-glycans in proteolytic processing and cell surface transport of the lassa virus glycoprotein gp-c date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: f prnpsu lassa virus glycoprotein is synthesised as a precursor (pregp-c) into the lumen of the endoplasmic reticulum. after cotranslational cleavage of the signal peptide, the immature gp-c is posttranslationally processed into the n-terminal subunit gp- and the c-terminal subunit gp- by the host cell subtilase ski- /s p. the glycoprotein precursor contains eleven potential n-glycosylation sites. in this report, we investigated the effect of each n-glycan on proteolytic cleavage and cell surface transport by disrupting the consensus sequences of eleven potential n-glycan attachment sites individually. five glycoprotein mutants with disrupted n-glycosylation sites were still proteolytically processed, whereas the remaining n-glycosylation sites are necessary for gp-c cleavage. despite the lack of proteolytic processing, all cleavage-defective mutants were transported to the cell surface and remained completely endo h-sensitive. the findings indicate that n-glycans are needed for correct conformation of gp-c in order to be cleaved by ski- /s p. lassa virus belongs to the family of arenaviridae which includes other important human pathogens like machupo virus, junin virus, guanarito virus and sabia virus as well as the prototype of this family, lymphocytic choriomeningitis virus (lcmv). lassa virus is endemic in certain parts of west africa and causes an estimated clinical cases of a systemic viral illness per year, with a mortality rate of - % [ ] . lassa fever is not only a public health concern in endemic areas, it is also sporadically exported to other parts of the world [ ] . lassa virus is an enveloped virus with a bi-segmented rna genome which encodes four viral proteins in an ambisense coding strategy: the glycoprotein precursor pregp-c, a nucleoprotein (np), the viral polymerase l and the matrix protein z (for review see [ ] ). the lassa virus glycoprotein is translated as an inactive precursor (pre-gp-c) into the lumen of the endoplasmic reticulum and is then cotranslationally cleaved into gp-c and a stable signal peptide [ ] . the latter one plays an important role in the subsequent posttranslational maturation cleavage of gp-c into its subunits gp and gp [ , ] . lassa virus gp-c is cleaved at the c-terminus of a non-basic amino acid residue of the cleavage motif r-r-l-l by the subtilase ski- / s p, which is unusual for fusiogenic glycoproteins of enveloped viruses [ ] . so far, only viral glycoproteins of the arenavirus family and the glycoprotein of the crimean-congo hemorrhagic fever virus belonging to the family of bunyaviridae are known to be cleaved by ski- / s p, an enzyme that normally plays a role in regulation of the lipid metabolism of the cell [ , [ ] [ ] [ ] . fusiogenic glyc-oproteins of most other enveloped viruses are proteolytically cleaved c-terminally of a single arginine residue or at a multibasic recognition motif by the subtilase furin [ ] . n-glycans play not only an important role in folding and intracellular transport of viral glycoproteins but also are known to modulate their antigenicity and activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . glycoproteins of arenaviruses vary considerably in number and position of potential n-glycosylation sites as shown by an n-glycosylation prediction program ( table ). the lassa virus glycoprotein gp-c contains eleven potential n-glycosylation consensus sites (n-x-s/t, in which x can be any amino acid except proline), where seven of these are located in the gp -and four in the gp subunit (table ) . using limited n-glycanase digestion, wright et al. [ ] demonstrated for lcmv gp that five potential glycosylation sites on gp- were utilized and two of the three sites on gp- . however, for lassa virus gp it has not yet been shown which sites are actually used and how n-glycosylation affects the characteristics of the glycoprotein in respect to cleavage and transport along the exocytotic pathway. to address this question, we investigated in the present report each potential n-glycosylation site of the lassa virus glycoprotein concerning n-glycan maturation, cleavage of gp-c and glycoprotein transport to the cell surface. we provide evidence that each of the n-glycosylation sites is used for n-glycan attachment. however, seven n-glycan mutants failed in their ability to get proteolytically processed, while n-glycosylation sites are individually dispensable for cleavage of gp-c. furthermore, we demonstrate that both uncleaved precursor and cleavage-defective n-glycan mutants are transported to the cell surface and remain mannose-rich. vero cells were cultured in dulbecco's modified eagle's medium (dmem, gibco) supplemented with % fetal calf serum (fcs), units/ml penicillin, and . mg/ml streptomycin. oligopeptide comprising the amino acids - of pregp-c was chemically synthesized and covalently linked to keyhole limpet hemocyanin (klh, pierce) as a carrier protein by the cross-linker agent n-[α-maleimidoacetoxy] succinimide ester (amas) and used for repeated immunization of rabbits as described before [ ] . the obtained antiserum rb-α-gp was tested by peptide standard elisa [ ] and used for immunoblot analyses. the full length glycoprotein of lassa virus (strain josiah) was expressed using the beta-actin promotor-driven pcaggs vector [ , ] . lassa virus n-glycosylation mutants were generated by recombinant pcr using overlapping oligonucleotides, which will be made available on request. sequences of mutants were confirmed by dna sequencing. vero cells were transfected with wild type and mutated recombinant dna using lipofectamine (gibco/invitrogen). lassa virus pregp-c contains potential n-glycosylation sites, in the gp- -and in the gp- -subunit (fig. ) . to investigate whether each n-glycosylation site is used for n-glycan attachment, the corresponding serine or threonine residues of potential n-glycosylation motifs (n-x-s/ t) were individually changed to alanine. gp- mutants were designated t a, s a, t a, s a, s a, s a and t a and gp- mutants s a, s a, s a and s a. all cdna constructs were expressed transiently in vero cells using the beta-actin promoterdriven mammalian expression vector pcaggs. western blotting was performed using antiserum directed against the c-terminus of gp- . as shown in fig. a (lanes - , upper panel) and fig. b (lanes - , upper panel), all mutants showed faster migration on a sds gel compared to wild-type gp-c indicating that each n-glycosylation site is used for n-glycan attachment. next we determined the effect of the individual n-glycans on the maturation cleavage by analysing the presence of the cleaved subunit gp- . disruption of the n-glycosylation sites within the gp- mutants s a, s a and t a has no influence on gp-c cleavage ( fig. a, lanes , and , lower panel) . in contrast, the lack of n-glycan attachment of the gp- mutants t a, s a, s a and s a abolished the proteolytical processing by ski- /s p ( fig mutants s a and s a revealed significantly faster electrophoretic mobility than wild type gp- ( fig. b ; lanes - , lower panel) showing that the respective n-glycosylation sites are used for n-glycan attachment. n-glycosylation has long been known to be essential for correct folding and intracellular transport of viral glyco-proteins, as shown for instances for influenza hemagglutinin [ ] . in order to investigate the importance of the individual n-glycosylation sites of lassa virus gp-c for intracellular trafficking we analysed cell surface expression of the mutated glycoproteins using a biotinylation approach. transfected vero cells were labelled with nonmembrane-permeating biotin as described in material and methods. after biotinylation, cells were lysed and gp-proteolytic processing of n-glycosylation mutants figure proteolytic processing of n-glycosylation mutants. a vero cells expressing either wild-type gp-c or gp- n-glycosylation mutants t a, s a, t a, s a, s a, s a and t a. b vero cells expressing either wild-type gp-c or gp- n-glycosylation mutants s a, s a, s a and s a. the expressed proteins were separated by sds-page on % acrylamide gels and immunoblotted. non-cleaved gp-c and its cleaved form gp- are detected by immunostaining using the antiserum rb-α-gp- . c and gp-c n-glycan mutants were immunoprecipitated using polyclonal antiserum directed against the c-terminus of gp-c. the precipitates were separated on an sds gel and transferred to nitrocellulose. surface-expressed glycoprotein protein was then visualised with a streptavidin-peroxidase complex. fig. a (lanes - ) and b (lanes - ) demonstrate that all gp- and gp- n-glycosylation mutants are transported to the cell surface in a similar manner compared to wild-type gp indicating that a loss of a single n-glycosylation attachment site and proteolytic cleavage are not necessary for cell surface transport of gp. for lcmv it was shown that maturation cleavage deficient mutants are efficiently transported to the cell surface [ ] . both cleavage and cell surface expression of lcmv gp-c are impaired in parallel either when no n-glycosylation occurs or a proline residue is present at position [ , ] . since many cleavage deficient mutants have been shown to exhibit regular cell surface transport, the two reported cases in which cell surface expression was abolished might be due to misfolding in the endoplasmic reticulum with subsequent degradation [ , ] . cell surface transport of gp-c n-glycosylation mutants figure cell surface transport of gp-c n-glycosylation mutants. vero cells were transfected with gp- (a) and gp- (b) n-glycosylation mutants of lassa virus glycoprotein. at h post-transfection, cells were surface labelled with biotin. following cell lysis and immunoprecipitation using α-gp- , samples were subjected to sds-page and blotted to nitrocellulose. surface biotin-labelled proteins were visualized with streptavidin-peroxidase and chemiluminescence. lcmv and lassa virus gp-c are cleaved by the same protease, ski- /s p, although proteolytic processing of the gps occurs in different subcellular compartments. lassa virus gp-c is cleaved early along the secretory pathway in the er or an early golgi stack, whereas lcmv gp-c is cleaved in the golgi or post-golgi compartment [ , , ] . furthermore, for lcmv gp-c it has been shown that transition of mannose-rich carbohydrates to complex carbohydrates occurred before maturation cleavage [ ] . in order to analyse the glycosylation status of lassa virus glycoproteins on the outer cell membrane, cell surface biotinylation was combined with a subsequent detachment of mannose-rich n-glycans by digestion with endo h or removal of complex n-glycans using pngase f (fig. ) . the gp- subunit is detectable at the cell surface and displays partial endo h-resistance confirming a modification of its carbohydrate decoration during glycoprotein transport to the cell surface (lane ). interestingly, the glycoprotein precursor gp-c which is transported to the cell surface remains completely endo h-sensitive suggesting that cleavage might be a prerequisite for further complex glycosylation. in addition, uncleaved endo hsensitive lassa virus gp-c is also transported to the cell surface of ski- /s p deficient cells (data not shown). the finding that only an endo h-sensitive form of lassa virus gp-c was found at the cell surface suggests that partial endo h-resistance of the lassa virus glycoprotein is only acquired when the glycoprotein is cleaved into its subunits. this observation is in contrast to findings for the lcmv glycoprotein where an endo h-resistant form of gp-c was described [ ] . however, since cleavage of lassa virus gp-c by ski- /s p occurs earlier in the exocytotic pathway than lcmv gp-c [ , , ] , cleavage of lassa virus gp-c occurs prior to trimming of the mannose-rich n-glycans to complex sugar types. it can not be ruled out that the use of glycosylation sites and the n-glycan trimming differs between recombinant expressed glycoproteins and gp in lasv-infected cells. it was shown recently that recombinant expressed m protein of sars-cov gained complex-type n-glycosylation whereas in sars-cov infected cells n-glycosylation of m remained endo h-sensitive [ ] . however, in general a good correlation between results obtained after solitary expression and studies using the homologues viral system have been observed. to address this kind of issues, the development of a "reverse genetic system" for lasv will be necessary that would allow analysing the role of glycosylation of gp in the correct viral context. taken together, our study suggest that individual n-linked oligosaccharides of the lassa virus glycoprotein differ greatly in terms of their importance for correct protein folding which seems to be important for activation cleavage by ski- /s p. the differences between the precursor glycoprotein gp-c and the cleaved subunits gp- and gp- , not only with respect to n-glycosylation, remain an enigma since only the cleaved glycoprotein subunits are incorporated into virus particles [ ] . lassa fever imported lassa fever in germany: molecular characterization of a new lassa virus strain lassa virus signal peptide of lassa virus glycoprotein gp-c exhibits an unusual length identification of lassa virus glycoprotein signal peptide as a trans-acting maturation factor the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski- /s p identification of a novel consensus sequence at the cleavage site of the lassa virus glycoprotein characterization of the glycoproteins of crimean-congo hemorrhagic fever virus crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski- endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase ski- /s p host cell proteases controlling virus pathogenicity individual roles of n-linked oligosaccharide chains in intracellular transport of the paramyxovirus sv fusion protein folding and assembly of viral membrane proteins influence of n-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein importance of hemagglutinin glycosylation for the biological functions of influenza virus structure-based, targeted deglycosylation of hiv- gp and effects on neutralization sensitivity and antibody recognition influence of n-glycans on processing and biological activity of the nipah virus fusion protein carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size role of conserved glycosylation sites in maturation and transport of influenza a virus hemagglutinin functional analysis of the individual oligosaccharide chains of sendai virus fusion protein n-glycans attached to the stem domain of haemagglutinin efficiently regulate influenza a virus replication post-translational processing of the glycoproteins of lymphocytic choriomeningitis virus efficient selection for highexpression transfectants with a novel eukaryotic vector recombinant expression of lymphocytic choriomeningitis virus strain we glycoproteins: a single amino acid makes the difference characterization of severe acute respiratory syndrome coronavirus membrane protein we thank e. lecompte, m. eickmann and h.-d. klenk for helpful discussions and p. neubauer-rädel for excellent technical assistance. this work was supported by the deutsche forschungsgemeinschaft, sachbeihilfe ga / - , sfb tp , and the graduiertenkolleg protein function at the atomic level. the author(s) declare that they have no competing interests. re and ts carried out experiments, participated in the analysis of the results and drafted the manuscript. ol helped to draft the manuscript and revised it critically. wg designed the study, participated in the analysis of the results and helped to draft the manuscript. all authors read and approved the final manuscript. key: cord- -fj l che authors: bragstad, karoline; nielsen, lars p; fomsgaard, anders title: the evolution of human influenza a viruses from to : a complete genome study date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: fj l che background: knowledge about the complete genome constellation of seasonal influenza a viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. few complete genome sequences of influenza a viruses from europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza a viruses. we have studied the evolution of circulating human h n , h n and h n influenza a viruses from to , we analysed danish human influenza a viruses and characterised complete genomes. results: h n was the prevalent strain in denmark during the study period, but h n dominated the – season. h n viruses were first observed in denmark in – . after years of little genetic change in the h n viruses the – season presented h n of greater variability than before. this indicates that h n viruses are evolving and that h n soon is likely to be the prevalent strain again. generally, the influenza a haemagglutinin (ha) of h n viruses formed seasonal phylogenetic clusters. different lineages co-circulating within the same season were also observed. the evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the fujian-like viruses in – . also evolutionary stasis-periods were observed which might indicate well fit viruses. the evolution of h n viruses have also been influenced by gene reassortments between lineages from different seasons. none of the influenza genes were influenced by strong positive selection pressure. the antigenic site b in h n ha was the preferred site for genetic change during the study period probably because the site a has been masked by glycosylations. substitutions at ctl-epitopes in the genes coding for the neuraminidase (na), polymerase acidic protein (pa), matrix protein (m ), non-structural protein (ns ) and especially the nucleoprotein (np) were observed. the n-linked glycosylation pattern varied during the study period and the h n isolates from to were highly glycosylated with ten predicted sequons in ha, the highest amount of glycosylations observed in this study period. conclusion: the present study is the first to our knowledge to characterise the evolution of complete genomes of influenza a h n , h n and h n isolates from europe over a time period of seven years from to . more precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition. every year the influenza a virus causes human infection with varying severity depending on the host acquired immunity against the particular virus strain. three to five million people experience severe illness and . to . million people die of influenza yearly worldwide (who eb / ). the influenza virus evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (ha) and neuraminidase (na) or by reassortment of segments from different viruses co-infecting the same cell leading to a new stain with a ha (and na) not seen in the population before (shift). in the worst case, shifts may cause pandemics. there have been three pandemics the last hundred years, the spanish flu in (h n ), the asian flu in (h n ) and the hong kong flu in (h n ). it is believed that new pandemics emerge through shifts with strains from the avian reservoir, as was the case of the pandemics of and , or by direct introduction of an avian strain into the human population as suggested for the pandemic [ ] . at present only two of the possible ha subtypes (h and h ), and two of the nine possible na subtypes (n and n ) are circulating in man. h n and h n influenza a viruses have co-circulated in the human population since the re-emergence of h n in , increasing the possibility for genetic reassortments. the prevalence of the different subtype combinations may vary from season to season. the h n has been the predominant influenza a strain during the last years, with the exception of the - and - seasons where h n infections dominated [ ] . in the - season a new reassorted human strain, h n , emerged in europe and became established in the autumn [ , ] . the new h n subtype was covered by the - h and n trivalent vaccine components and because both h and n viruses had circulated the previous years some degree of herd immunity against the new strain was expected. the h n viruses were not associated with severe influenza illness that season. in , a new lineage a/fujian/ / (h n )-like emerged in asia and caused significant outbreaks on every continent [ , ] . for the northern hemisphere the who issues the recommendation for strains to be included in the trivalent vaccine for the next season based on epidemiological data and antigenic and genetic analyses of circulating strains. until the recent release of over , complete influenza a genome sequences from the influenza genome sequencing project managed by us national institute of allergy and infectious diseases [ , ] the relative prevalence of influenza virus varies from season to season. influenza a h n was the dominating strain in denmark during the last seven years, with the exception of the - season where the h n viruses dominated, as can be seen in figure . based on phylogenetic analysis of the ha and na nucleotide sequences from to (figure ), ten isolates representative for the phylogenetic clustering of sequences from each subtype in each season, as far as possible, were included in the final ha and na tree ( figure ) and representatives were chosen for complete genome sequencing. generally the h n ha and na genes formed seasonal phylogenetic clusters ( figure ). however, we observed that strains of different lineages and clusters cocirculated within the same season and that viruses had reassorted with viruses from previous seasons ( figure ). the ha gene of the influenza h n strains from the - season formed a phylogenetic subclade to a/ moscow/ / (h n ) and a/sydney/ / (h n ) (represented by a/memphis/ / ) (figure ), located between a/moscow/ / and a/panama/ / (not shown). the antigenicity of these strains was a/moscow/ / (h n )-like in a haemagglutination inhibition assay, and will therefore be referred to as moscow-like throughout this report. the na and the internal genes were all a/moscow/ / (h n )-like, with the exception of the matrix (m) gene that clustered as a subclade to the a/new york/ / -like strains ( figure ). figure ). thirteen isolates from this season were available for sequencing, and all were of the h n subtype. these sequences represented two different co-circulating lineages (figure ). lineage i is a/bayern/ / (h n )-like and lineage ii include the h n strains of today and the a/new caledonia/ / (h n ) vaccine reference strain (figure ). the phylogenetic trees of na and the internal genes showed the same topology ( figure and ) . the lineage ii strains are characterised by a deletion k in ha (k in h numbering) ( table ) * amino acids in brackets indicate less than half but more than two substitutions at the given amino acid position within a season. a single amino acid change in one position is not shown. amino acids separated by '/' indicate equal substitutions of either amino acid at the given position. letters in upper case above an amino acid indicate the antigenic site location of the residue. in n the upper case letter ' p ' stands for phylogenetically important region (pir) and the following letters indicate the actual pir. based on the ratio of non-synonymous versus synonymous substitutions none of the influenza a genes were directly influenced by positive selection (dn/ds< ) (table ) . however, as expected the ha region of both h and h viruses were more influenced by evolutionary pressure (table ) . we applied fel and slac maximum-likelihood methods to estimate individual positively selected sites in h n ha and na and added rel for smaller datasets in all genes (se methods section). the fel method found one site in the h protein (n = ), position (p = . ) to be positively selected, while the more conservative slac analysis found none. no positive selected sites were predicted for the n genes (n = ) and none in the internal genes (n = ) estimated by fel and slac. the rel analysis retrieved four sites in the m gene to be selected namely positions , , and . no sites in ha (n = ) and na (n = ) of the h n viruses were directly positively selected with any of the three methods of analysis. [ ] and it was anticipated that there would be some extent of herd immunity in the population against this new reassortant. the phylogenetic trees of h n ha and na showed seasonal clusters but also co-circulating lineages within seasons. the introduction of the a/fujian/ / (h n ) strains in - caused a "jump" in the evolution of both ha and na genes. many of the substitutions in ha introduced with the a/fujian/ / (h n )-like viruses have become fixed, probably reflecting a very fit virus. the genetic variation before the - season may have been more influenced by introduction of new viruses through viral migration than adaptive evolution of the genes. a constant rate of drift was not observed for ha but instead periods of change followed by stasis periods. the low dn/ds ratios ( . for ha and . for na) also indicated that the influenza genes were not directly influenced by positive selection. reassortments between co-circulating strains and viruses from previous seasons, and introduction of viruses from other parts of the world might play a larger role than natural selection for some seasons, as also observed by others [ , ] . [ , , , ] . the reassorted h n viruses possessed only the ha from a/new caldonia/ / (h n )-like viruses and the rest of the genome from a/ moscow/ / (h n )-like viruses as also reported by others using partial sequences [ ] . the h n viruses have been introduced to denmark from elsewhere and are not a local reassortant. (table ) suggesting they may be important for viral escape from the host immune system and the overall fit of the virus. the a/fujian/ / -like(h n ) viruses did not antigenically match the a/moscow/ / (h n ) strain included in the - vaccine [ , ] . the ha substitution d n was however not responsible for the antigenic drift of the fujian-like viruses. it has been shown that only two amino acid changes, h t and q h, specified the antigenic difference from moscow-like to fujian-like [ ] , both are located at antigenic site b. the t and h amino acids have been maintained in all danish isolates after the introduction of the fujian-like viruses. with the a/california/ / (h n )-like viruses in - , site has changed from k to s in some isolates and to n in others. s has been found at this position before and n k was the only cluster-difference substitution between isolates from the seasons / and / [ ] . it was therefore suggested that substitutions at this site alone could have large antigenic effect. antigenic site a, located in a loop, makes few contacts with the rest of the structure, therefore and may change drastically without influencing on the overall shape of the ha molecule. antigenic site a is supposed to be ideal for antibody binding and for amino acid replacements [ ] . the preferred antigenic site in the change from a/moscow/ / (h n ) to a/fujian/ / (h n )-like viruses was site b. this observation is in accordance with previously published data [ ] . one region in ha, position to , that influence antigenic site d, has changed drastically during the study period from gvs → dvs → dip → nip. the influence of antigenic site d was first apparent in the antigenic change from fujian-like viruses to california-like viruses; however, the antigenic site b was still the preferred site for antigenic change. it has been proposed that a minimum of four substitutions in two or more antibody binding sites are required for an epidemically important strain [ ] . gulati et al., [ ] [ , [ ] [ ] [ ] [ ] . the substitution h y is the only neuraminidase resistance mutation identified in h n viruses to date [ , ] . danish isolates did not possess any resistance mutations in the nas. neuraminidase inhibitory drugs are rarely used for influenza prophylaxis or treatment in denmark. t-cell epitopes are more conserved than antibody epitopes. fifteen per cent of the t-cell epitopes are conserved while only . % of the antibody epitopes [ ] . the reason for this higher degree of conservation is that % of the antibody epitopes are located in the variable glycoproteins ha and na, while only % of the t-cell epitopes are found in these proteins [ ] . recent research has shown some degree of escape from ctl-mediated immunity in addition to escape from neutralizing antibodies [ ] . in our dataset we found several substitutions in regions involved in protective t-cell response [ ] in na, pa, m , ns and most in the np protein. this is not unexpected because most t-cell epitopes are defined for the np protein and this protein is the main target for the cytotoxic host immune response [ , ] . the extensive variations in the t-cell epitopes during to suggest that these regions and the antibody epitopes are working together for efficient escape from the host defence responses. the m proteins from the danish wisconsin-like viruses in - possessed the substitution s n, associated with resistance to matrix inhibitory drugs, like amantadine [ , , , ] . these types of drugs are not used for prophylaxis or treatment in denmark. the s n substitution is therefore not a local introduced resistance mutation. we cannot exclude that this substitution has arisen by chance, but it is more likely that the mutation has emerged as a resistance mutation in other countries like the usa [ ] and australia [ ] where the prevalence of amantadine resistance is high. the resistance may also be related to the increased use of this drug in asia during the sars epidemic [ ] . the a/fujian/ / (h n )-like clinical danish viruses had several substitutions in ha at sites that might influence the virus' capability for egg growth [ , ] . these include; a t, i n, h t, q h, w r and g d. the a/california/ / (h n )-like viruses had further changes at positions k s/n, y f, s f and v i and a/wisconsin/ / (h n ) possessed in addition s f and d n. all isolates after the - a/moscow/ / (h n )-like viruses possess s g. in recent years h n viruses have had poor replication efficiency in eggs [ , ] . it has been shown that positions , and are critical determinants for egg growth. the changes g v and v i increased egg viral replica-tion of a/fujian/ / (h n ) viruses so did the changes g v and a t for a/california/ / (h n ) viruses [ , ] . on the contrary, others have stated that the v i change in combination with t and h do not result in viral recovery in eggs [ , ] . this might explain the delay in the - vaccine production for the northern hemisphere due to egg propagation difficulties with the a/wisconsin/ / (h n ) strain. the influence on replication efficiency by the other substitutions observed at receptor binding sites should be investigated further. we did not observe amino acids in the n na protein that would decrease virus replication in eggs. the amino acids known to give good replication in eggs (q , k and y ) [ ] were all present in this dataset. oligosaccharides at the surface proteins ha and na might have greater impact on viral escape from the immune system than single amino acid changes in the antigenic sites. oligosaccharides which are recognised as "self" by the host immune system may pose conformational changes in the molecule and mask antigenic sites, thereby prevent binding of host antibodies. the number of n-linked glycosylation sites in the h ha protein has increased during the years from only three attachment sites in [ , ] to ten predicted sites in the danish isolates after . the a/fujian/ / (h n )-like stains from the - season gained a potential glycosylation site at position , thereby masking the supposed "key" site for antigenic change [ ] . substitutions at antigenic site b and the predicted n-glycosylation at position in ha antigenic site a together with a stronger na might have contributed to the increased infectivity of the reassorted fujian-like viruses of the - season, causing an epidemic in denmark. we have shown that the preferred antibody epitope for genetic change is antigenic site b for the danish dataset also reflecting that site a is camouflaged by glycosylation. thus the antigenic changes at a glycosylated site a may not play a major role in escape from the immune system as long as the glycosylation is present. six potential n-glycosylation sites have been conserved in the n na danish dataset from to . the majority of isolates from to have lost the site at position which is located in a ctl epitope (hla-a* ) region of na [ ] . the recent nas may therefore be more easily recognised by the cytotoxic immune system. we found two predicted n-glycosylation sites ( and ) in the n na stalk region in sequences from - . greater density of carbohydrate in the stalk region of na might reflect a need for proteolytic protection. the two observed carbohydrates in the stalk have been reported for other isolates in other time periods and other continents [ , ] . the stalk region has therefore stayed unchanged and the two sequons seem to be conserved. as expected a higher dn/ds ratio was observed for the surface glycoproteins, although none were directly influenced by positive selection. we observed that the ha region and the m protein have a slightly higher global dn/ds ratio than the other genes (table ). this is consistent with the findings of others [ , ] . the m protein is a membrane ion channel protein on the surface of the virus molecule, a higher dn/ds ratio for this protein compared to the internal proteins is expected. the ratio might, however, be biased because the m protein is spliced from m and the ds is suppressed for overlapping regions giving a higher dn/ds ratio [ ] . earlier findings support positive selection at sites involved in receptor and antigen binding [ , ] . five of the codons in ha proposed by bush et al., [ ] to be under positive selection were found to have changed in our ha dataset of h n since , namely: pos , , , and . in our h dataset (n = ) site was influenced by positive selection as calculated by fel analysis, but no positively selected sites were found applying the more conservative slac method. position may be involved in receptor binding and influence on the virus ability to grow in eggs [ ] . in a similar study on a slightly larger dataset (n = ) positions and were found to be positively selected [ ] . another study found positions and [ ] ; however, suggested positively selected sites may vary by the dataset applied, method used and the significance level selected for a site to be classified as positively selected. rel analysis identified four sites ( , , and ) in m under positive selection pressure. rel analysis tends to give better estimates on small datasets than slac and fel. sites , , and were still selected when the bayes factor cut-off was increased from to . further analysis would be needed to determine if these sites actually are positively selected. there is a need for complete genome analysis of european human influenza a viruses in order to gather a comprehensive picture of the evolution and migration of viruses. our results support the suggestion that the evolution of influenza a viruses is more complex than originally believed [ , ] . local short term evolution of influenza virus is a stochastic process, also involving gene reassortments. it will be interesting to further investigate how viruses from other parts of europe influence on the evolu-tion of danish isolates when more full length sequences from europe are made public. viral rna was extracted from µl of human nasal swab suspension or nasopharyngeal aspirate by qiaamp ® viral rna mini kit (qiagen, germany) as described by the manufacturer or by an automated magna pure lc instrument applying the magna pure lc total nucleic acid isolation kit (roche diagnostics, basel, switzerland). the different gene segments were amplified by onestep rt-pcr kit (qiagen) as previously described [ ] , applying a two minute elongation step for all genes. the primers for rt-pcr were segment specific but subtype universal targeting the highly conserved noncoding rna regions at the '-and '-end of each segment [ ] . pcr products were purified using the gfx™ pcr dna and gel band purification kit (amersham biosciences, germany) prior to sequencing. purified pcr products were sequenced directly. primer sequences are available upon request. the sequencing reaction was performed by abi prism ® bigdye™ terminators v . cycle sequencing kit (applied biosystems, usa) as described previously [ ] . the sequences were developed on an automatic abi prism ® genetic analyzer (applied biosystems) with cm capillaries. consensus sequences were generated in seqscape ® software v . (applied biosystems). sequence assembly, multiple alignment and alignment trimming were performed with the bioedit software v. . . [ ] . distance based neighbor joining (nj) phylogenetic trees and character based maximum parsimony (mp) trees were generated using the molecular evolutionary genetics analysis (mega) software v. . [ ] . maximum likelihood trees were generated by the phylogenetic analysis using parsimony (paup . ) software (sinauer associates, inc.) [ ] applying the hky nucleotide model, allowing transitions and transversions to occur at different rates. the between-seasons nucleotide distance means were computed as the arithmetic mean of all pair wise distances between two seasons in the inter-season comparisons using the mega v. . software [ ] . the global rate between dn and ds substitutions and the individual sitespecific selection pressure were measured by the likelihood based single likelihood ancestor counting (slac) method in datamonkey (modified suzuki-gojobori method) [ , ] . for datasets over sequences (h n = and n n = ) the hyphy package [ ] was applied. the estimations are likelihood-based, employing a codon model cross between hky and mg . to elucidate single, positively selected amino acids, the ha and na datasets were analysed with slac and a two rate fixed effects likelihood (fel) [ ] using a likelihood approach with neighbor joining phylogenetic trees (hyphy). sites with dn>ds with a < . significance level for likelihood ratio test (lrt) were implied as positively selected for the large ha and na datasets. for the small datasets (genes coding for the internal proteins < and the h n viruses < ) we additionally ran a random effects likelihood (rel) analysis using an empirical bayes approach with nj phylogenetic trees in datamonkey [ ] . this method is expected to calculate positively selected sites more accurately in small datasets. the accepted significance level for a positively selected site was set at < . (two-tailed binominal distribution) for slac and fel analyses and > bayes factor for rel. potential n-linked glycosylation sites were predicted using nine artificial neural networks with the netnglyc . server [ ] . a threshold value of > . average potential score was set to predict glycosylated sites. the n-glycosite prediction tool at los alamos [ ] was used to visualise the fraction of isolates possessing certain glycosylated sites along the sequence alignment. the specific measure of antigenic distance between two strains of influenza were calculated as p epitope values by the method suggested by muñoz, et al., [ ] . the p epitope value was calculated as the number of mutations within an antibody antigenic site divided by the number of amino acids defining the site. it is assumed that an antigenic epitope which has the greatest percentage of mutations is dominant, because that epitope is influenced by the greatest selective pressure from the immune system. the p epitope distance is defined as the fractional change between the dominant antigenic epitopes of one strain compared to another. the p epitope calculator [ , ] was applied for h sequences. residues in antigenic epitopes were collected from several references [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] , ] . influenza: the mother of all pandemics recent changes among human influenza viruses mild to moderate influenza activity in europe and the detection of novel a(h n ) and b viruses during the winter of - 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influenza season in the united states increased adamantane resistance in influenza a(h ) viruses in australia and neighbouring countries in hemagglutinin residues of recent human a(h n ) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes receptor-binding properties of modern human influenza viruses primarily isolated in vero and mdck cells and chicken embryonated eggs improvement of influenza a/fujian/ / (h n ) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics single amino acid substitutions in the hemagglutinin of influenza a/singapore/ / (h n ) increase virus growth in embryonated chicken eggs effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h n virus hemagglutinin variation and infectivity neutralization in influenza genetic variation in neuraminidase genes of influenza a (h n ) viruses long intervals of stasis punctuated by bursts of positive selection in the seasonal evolution of influenza a virus stochastic processes are key determinants of short-term evolution in influenza a virus positive selection on the h hemagglutinin gene of human influenza virus a genome characterisation of the newly discovered avian influenza a h n virus subtype combination universal primer set for the full-length amplification of all influenza a viruses new avian influenza a virus subtype combination h n identified in danish mallard ducks bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt integrated software for molecular evolutionary genetics analysis and sequence alignment phylogenetic analysis using parsimony datamonkey: rapid detection of selective pressure on individual sites of codon alignments a method for detecting positive selection at single amino acid sites hyphy: hypothesis testing using phylogenies not so different after all: a comparison of methods for detecting amino acid sites under selection prediction of n-glycosylation sites in human proteins tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin quantifying influenza vaccine efficacy and antigenic distance calculation of pepitope using microsoft excel location of antigenic sites on the three-dimensional structure of the influenza n virus neuraminidase the antigenic structure of the influenza virus a/pr/ / hemagglutinin (h subtype) structure of the catalytic and antigenic sites in influenza virus neuraminidase phylogenetically important regions of the influenza a h hemagglutinin protein the structure and function of the hemagglutinin membrane glycoprotein of influenza virus influenza virus neuraminidase: structure, antibodies, and inhibitors the neuraminidase of influenza virus. proteins we thank bente Østergaard, christine valdez and bente andersen for their excellent laboratory work and sofie e. midgley for kindly proof reading the manuscript. the author(s) declare that they have no competing interests. kb conceived and designed the experiments. kb performed the experiments, data analysis and wrote the paper. lpn and af contributed reagents and materials. afo supervised the research. further lpn and af critically revised the manuscript and af gave the final approval for publication. all authors read and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge key: cord- -tatae hs authors: lin, wenyao; fan, huiying; cheng, xiaoliang; ye, yu; chen, xiaowei; ren, tao; qi, wenbao; liao, ming title: a baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with h n avian influenza virus in mice date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: tatae hs background: avian influenza viruses of h n subtype have become highly prevalent in avian species. although these viruses generally cause only mild to moderate disease, they can infect a wide variety of species, including chickens, quail, turkeys, ducks, geese, pheasant, partridge, and pigeon, even transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. results: the results showed that stronger immune responses were induced in a mouse model immunized with bv-dual-ha than in those vaccinated with a dna vaccine encoding the same antigen. moreover, complete protection against lethal challenge with h n virus was observed in mice. conclusion: bv-dual-ha could be utilized as a vaccine candidate against h n virus infection. influenza a viruses of the h n subtype have become highly prevalent in poultry in many countries, and although these viruses generally cause only mild to moderate disease, they can infect a wide variety of species, including chickens, quail, turkeys, ducks, geese, pheasant, partridge, and pigeon [ ] [ ] [ ] [ ] . more importantly, occasional transmission of h n viruses from landbased poultry to humans and pigs have been reported [ ] [ ] [ ] . some investigations suggest that a significant proportion of h n field isolates have acquired human virus-like receptor specificity; a few that could recognize α , -linked sialic acid (saα - ) have been transmitted directly to humans [ ] [ ] [ ] [ ] . in addition to possessing human virus-like receptor specificity, avian h n viruses induce a typical human flu-like illness, which can easily go unreported, and therefore have the opportunity to circulate, reassort, and improve transmissibility [ , [ ] [ ] [ ] [ ] . hence, global concern is focused on the prevention and treatment of h n avian influenza virus infections. prevention of avian influenza is mainly through vaccination. currently, most avian influenza vaccines used in clinics are the inactivated type, which are propagated in embryonated chicken eggs. however, the use of inactivated avian influenza vaccines can induce little or no cellular immune response; thus, it cannot provide wide and persistent protection against influenza, and it will interfere with serological monitoring. in addition, eggbased influenza vaccine production is dependent on the availability of embryonated eggs, which is at risk in the event of outbreaks of avian diseases. in view of these potential drawbacks, we sought to develop a new type of h n vaccine using the baculovirus dual expression system constructed in this study. the baculovirus autographa californica multiple nucleopolyhedrovirus (acmnpv) has traditionally been an excellent tool to overexpress recombinant proteins in insect cells. since the discovery that baculovirus is capable of entering mammalian cells and mediating transgene expression under the promoter active in mammalian cells [ ] , baculoviral vectors have been exploited as versatile vaccine vehicles to produce vaccine candidates against different pathogens. recently, acmnpv has been further engineered for application as a new eukaryotic display system to express foreign proteins on the surface of the viral envelope [ ] [ ] [ ] [ ] [ ] and formed a hedgehog-shaped "fake virus". this display system relies on the main envelope protein of acmnpv gp protein, which causes the surface display of foreign proteins on the baculovirus surface. this method has been extended to develop pseudotype baculoviruses as a potential vaccine delivery platform. several research groups have demonstrated that direct vaccination with this kind of pseudotype baculoviruses can induce high titers of antigen-specific antibodies [ , ] . subsequently, it was determined that some natural viral envelope proteins such as influenza hemagglutinin (ha) can be displayed on the baculovirus surface, even without fusion with gp . tang et al. [ ] and yang et al. [ ] reported that signal peptide (sp) and cytoplasmic tail (ct) domains of gp can enhance the display of ha on the viral surface, while the transmembrane (tm) domain of gp impairs ha display. therefore, a chimeric ha with sp and ct derived from gp was chosen for our study. combining the characteristics of baculovirus as a gene delivery vehicle and surface display system, we constructed a "baculovirus dual expression system," (bv-dual-ha), which is capable of displaying h n -ha protein on the surface of the viral envelope and expressing it upon transduction in mammalian cells. the main objectives of this study were: i) to effectively display functional ha on the baculoviral envelope in the hope that ha would retain superior immunogenicity upon in vivo immunization, and ii) to efficiently express ha in transduced mammalian cells. the results showed that stronger immune responses were induced in a mouse model immunized with bv-dual-ha than in those vaccinated with a dna vaccine encoding the same antigen. moreover, complete protection against lethal challenge with h n virus was observed in mice, indicating the potential of bv-dual-ha as a vaccine candidate against h n virus infection. six-week-old balb/c female mice were purchased from southern medicine university, guangzhou, china, and were housed, fed in microisolator units according to the veterinary guidelines of south china agricultural university and all animal experiments were approved by the south china agricultural university institutional animal care and use committee. a porcine kidney pk- cell line free of pcv contamination (atcc ccl ) was maintained in dulbecco's modified eagle's medium (invitrogen, usa) and supplemented with % (v/v) heat-inactivated fetal bovine serum (fbs; invitrogen, usa), μg/ml of streptomycin, and iu/ml of penicillin. the spodoptera frugiperda cells (sf ) used to propagate wild-type and recombinant baculoviruses were cultured in grace's insect media (gibco; invitrogen, usa) and supplemented with % heat-inactivated fbs at °c. mdck cells were used to propagate avian influenza virus and were cultured in minimal essential medium (mem) containing % fetal calf serum at °c. the low-pathogenicity influenza a/chicken/guangzhou/v/ (h n ) virus was isolated in guangdong province, china. it is a mouse-adapted h n strain that can cause infection and death in mice, and was used to challenge immunized mice in this study. to construct a baculovirus transfer vector, psurf-ha, a gene lacking the n-terminal sp and c-terminal ctd but encoding the ectodomain of ha, was amplified from pc-ha plasmid (a dna vaccine was constructed with the pcdna . (+) vector expressing h n ha protein in our lab; invitrogen) and inserted between the sequences encoding the gp signal peptide and gp cytoplasmic domains of a pbacsurf- vector (whose gp ectodomain and transmembrane domain were deleted earlier; novagen). to construct pcmvsurf-ha, a . -kb fragment of a chimeric ha gene was excised from psurf-ha by digestion with nhei and hindiii, and inserted into the nhei/hindiii sites of the pcdna . (+) vector (invitrogen). the cassette consisted of the cmv-ie promoter; chimeric ha gene was amplified by pcr and subcloned into a pfastbac™ plasmid with sali/hindiii treatment to generate bv-dual-ha. the resultant bv-dual-ha plasmid thus contained separate ha genes driven by cmv-ie and pph promoters. recombinant baculovirus bv-dual-ha was produced using the bac-to-bac ® system and was propagated in sf insect cells according to standard methods. virus particles were purified by rounds of sucrose gradient ultracentrifugation following standard protocols [ ] , and infectious titers were determined with the bd bacpak baculovirus rapid titer kit (clontech laboratories, usa). purified virus particles were absorbed onto carboncoated copper grids and incubated with a monoclonal antibody (mab) against ha of h n (prepared in our lab) for h. the grids were incubated with goat antimouse igg labeled with -nm gold particles (sigma) for min. after additional pbs washes, the grids were stained with % phosphotungstic acid (sigma, st. louis, mo, usa) and examined under transmission electron microscopy (h- ; hitachi, tokyo, japan). pk- cells were seeded at a concentration of . × cells/well into -well tissue culture plates (corning costar co., cambridge, ma, usa) and transduced with purified baculovirus particles at an moi of . after h incubation, the cells were fixed with absolute methanol for min at - °c, rinsed with pbs, and blocked with % bovine serum albumin for min at °c. the cells were then incubated with the primary anti-body (anti-ha mab) for h at °c, followed by pbs washes. the cells were then incubated with the secondary antibody (fitc-conjugated rabbit anti-mouse igg; sigma) for h at °c, followed by pbs washes. fluorescence images were examined under an inverted fluorescence microscope (olympus ix ). six-week-old balb/c female mice were randomly divided into four groups, each containing mice. three groups were vaccinated intramuscularly (i.m.) with pfu of bv-dual-ha, pfu of wild-type acmnpv (acmnpv-wt), and μg of pc-ha, respectively. on days and . the final group was used as the control and injected with μl pbs. serum samples were collected on days and for serological tests. on day , the mice were challenged intranasally (i.n.) with mld ( % mouse lethal dose) of influenza a/chicken/guangzhou/v/ (h n ) and observed for clinical signs over a -day period. mice were weighed daily and examined for disease. mice that lost more than % body weight were humanely euthanized. six days after challenge, mice from each group were sacrificed and the lungs, brains, livers, kidneys, and spleens were harvested to examine virus replication in spf embryonated eggs. the viral titer, expressed as eid ( % egg infection dose), was calculated by the reed-muench method. the hemagglutination inhibition (hi) assay and virus neutralization (vn) assay were performed as described previously [ ] . an analysis of variance and student's t-test were used to evaluate potential differences among the different groups with regard to the humoral immune responses, viral burdens, and body weights. differences between groups were considered significant at p < . . construction of bv-dual-ha is shown in figure ; bv-dual-ha harbored a gene cassette that consisted of the gp signal peptide, ha ectodomain gene, ha tm, ctd derived from gp , and poly(a). the dual promoter that consisted of the cmv immediate early enhancer-promoter and the polyhedrin promoter drove expression of the gene cassette. thus, a chimeric ha protein was designed to express both proteins on the viral envelope and in mammalian cells. as shown in figure a , the ha protein expressed with bright fluorescence could be detected by ha mab in bv-dual-ha transduced cells, but not in cells transduced with acmnpv-wt ( figure b ), indicating that bv-dual-ha can enter mammalian cells efficiently and express the ha protein. to verify that the ha protein was displayed on the viral envelope, purified viral particles were analyzed by immunoelectron microscopy. as shown in figure c , specific immunogold particles were evident on the surface of bv-dual-ha, indicating the incorporation of chimeric ha and their display on the baculoviral envelope, whereas no gold particles were observed on the surface of acmnpv-wt ( figure d ). moreover, incorporation of the chimeric ha did not alter virus morphology. immunization with bv-dual-ha induced the highest levels of h -specific hi antibodies and vn antibodies. at days after primary immunization, all the mice immunized with bv-dual-ha developed detectable h specific hi ( figure a ) and vn antibodies ( figure b ), while the titers were very low in mice immunized with pc-ha. following a booster immunization, the mean titers of hi and vn antibodies increased greatly in mice immunized with bv-dual-ha, and were significantly higher than those of the mice vaccinated with pc-ha (p < . ). to evaluate the potency of the recombinant baculovirus bv-dual-ha against lethal influenza virus challenge, the immunized mice were challenged with mld of h n v strain on day . starting from day post-challenge, the mice immunized with pc-ha, acmnpv-wt, or pbs displayed serious weight loss ( figure a ) and to determine the virus titers in tissues, mice in each group were sacrificed on day post-challenge, and their lungs, brains, kidneys, and spleens were collected for analysis. as shown in table , there were higher levels of virus titers in the lungs of the mice immunized with acmnpv-wt or pbs, and virus titers were slightly lower in the mice immunized with pc-ha. compared with those of the pc-ha group, no virus was detected in the lungs of mice immunized with bv-dual-ha. the viral titers in the brains, kidneys, and spleens were also analyzed, but they were much lower than that in the lungs. in the present study, we developed a baculovirus dual expression system that possesses a gene cassette consisting of the chimeric ha gene under control of the cmv-polyhedrin dual promoter. we also investigated the efficacy of bv-dual-ha as an avian influenza vaccine. our results clearly show that immunization with bv-dual-ha provided % protection, compared to the . % and % protection observed with pc-ha and acmnpv-wt immunization, respectively. after challenge, viral titers in the lungs, brains, kidneys, and spleens were determined on day post-challenge. we found that all mice vaccinated with bv-dual-ha had undetectable viral titers in these tissues, suggesting that antibodies induced by bv-dual-ha conferred sterilizing immunity. most mice vaccinated with pc-ha, acmnpv-wt, or pbs had detectable lung virus titers a b figure antibody responses in immunized mice with bv-dual-ha. mice were immunized i.m. with bv-dual-ha, pc-ha, acmnpv-wt or pbs. an identical booster immunization was carried out weeks later. serum samples were collected on day (red bars) and day (blue bars) to determine the hi antibody titers (a), vn antibody titers (b). all data represent the mean ± s.d. by day post-challenge. from these results, it is obvious that immunization with bv-dual-ha can induce a robust antibody response and confer complete protection against lethal virus challenge in a mouse model, indicating that bv-dual-ha is potential candidate vaccine that can prevent and control the pandemic spread of the h n influenza virus. activate the ha-specific immune reactions via the major histocompatibility complex ii-mediated antigen presentation pathway, leading to more potent immune responses. moreover, compared with the dna vaccine, the baculovirus can directly transduce apcs [ , ] resident in the muscle tissues, especially more efficient antigen presentation to dendritic cells (dcs), which are the most important apcs [ ] . furthermore, martyn et al. [ ] reported that transduction by a recombinant baculovirus was more efficient than the transfection of conventional dna plasmids driven by the cmv promoter in both cell lines and primary cells. the transduction of primary marmoset hepatocytes with recombinant baculovirus was times more efficient than dna transfection, highlighting a major advantage of recombinant baculovirus for the delivery of foreign genes to mammalian cells. in this study, i.m. immunization of acmnpv-wt alone provided % protection from the h n influenza lethal challenge. this might be because the baculovirus envelope protein gp recognizes the tlr molecule and thus activates an innate immune response [ ] . this is consistent with a previous study that determined that intranasal immunization with wild-type baculovirus alone also provides sufficient protection from the h n influenza lethal challenge [ ] , though we didn't evaluate the cellular immune responses. and there are accumulated studies have demonstrated that the baculovirus itself has the ability to induce innate immune responses through a signaling pathway that is dependent on toll-like receptor (tlr )/myd , which results in the production of various cytokines, including members of the ifn family [ , , ] . hervas-stubbs reported that baculoviruses have strong adjuvant properties in mice, promoting potent humoral and cd + t cell adaptive responses against co-administered antigens [ ] . besides, previously studies also have proved that baculovirus expressing prrsv gp and m protein [ ] , pcv cap protein [ ] , h n virus ha protein [ ] , pseudorabies virus glycoproteins [ ] could induced a high level of ifn-g responses. the unique ability of the baculovirus to induce innate and adaptive immunity may have contributed to the protection from h n influenza lethal challenge. accumulating evidence has shown that the h n virus has undergone extensive reassortment, and novel genotypes have continued to emerge and evolve into several clades; this may increase the likelihood of avian-to-human interspecies transmission [ , ] . thus, an ideal vaccine against the h n avian influenza virus should overcome the antigenic variability of the virus. the baculovirus vector contains a large genome that enables insertion of large, foreign dna fragments or the construction of multivalent vaccines. in subsequent studies, we plan to improve the protective range of this system using appropriate selection of ha genes derived from different clades of the h n avian influenza virus for the composition of multivalent vaccines. in addition, we are also ready to co-express ha proteins and other immunogenic proteins of the h n influenza virus, such as neuraminidase and m , in order to enhance the immune response and protective efficiency. our study provides an alternative method of applying the baculovirus dual expression system as an immunizing reagent against the influenza virus. considering its safety and cost-effectiveness, a simple scale-up would be sufficient to produce a high-titer recombinant baculovirus, enabling bv-dual-ha to be utilized as an alternative strategy to prevent and control the pandemic spread of the h n influenza virus. table virus titration results of different tissue on day post-challenge (log eid ) a virus titration results of different tissue on day post-challenge (log eid ) b isolation and characterization of prevalent strains of avian influenza viruses in china molecular characterization of h n influenza viruses: were they the donors of the "internal" genes of h n viruses in hong kong? a review of avian influenza in different bird species avian influenza virus: of virus and bird ecology human infection with influenza h n discovery of men infected by avian influenza a (h n ) virus cocirculation of avian h n and contemporary "human" h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? characterisation of a human h n influenza virus isolated in hong kong continuing evolution of h n influenza viruses in southeastern china human infection with an avian h n influenza a virus in hong kong in characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? evolution and molecular epidemiology of h n influenza a viruses from quail in southern china a novel genotype h n influenza virus possessing human h n internal genomes has been circulating in poultry in eastern china since genotypic evolution and antigenic drift of h n influenza viruses in china from efficient gene-transfer into human hepatocytes by baculovirus vectors baculovirus surface-displayed hemagglutinin of h n influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity baculovirus surface display of e envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model a baculovirus dual expression system-based malaria vaccine induces strong protection against plasmodium berghei sporozoite challenge in mice avian influenza virus hemagglutinin display on baculovirus envelope: cytoplasmic domain affects virus properties and vaccine potential hemagglutinin displayed baculovirus protects against highly pathogenic influenza baculovirus surface display of sars coronavirus (sars-cov) spike protein and immunogenicity of the displayed protein in mice models baculovirus expression vectors: a laboratory manual new york and wh freeman and co immunization with reverse-genetics-produced h n influenza vaccine protects ferrets against homologous and heterologous challenge involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus baculovirus induces type i interferon production through toll-like receptor-dependent and independent pathways in a cell-type-specific manner baculovirus-based vaccination vectors allow for efficient induction of immune responses against plasmodium falciparum circumsporozoite protein transient and stable expression of the hcv envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus host innate immune responses induced by baculovirus in mammals baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice insect baculoviruses strongly potentiate adaptive immune responses by inducing type i ifn construction and immunogenicity of pseudotype baculovirus expressing gp and m protein of porcine reproductive and respiratory syndrome virus construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type in mice a pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with h n avian influenza virus in mice and chickens new baculovirus recombinants expressing pseudorabies virus (prv) glycoproteins protect mice against lethal challenge infection submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution authors' contributions wyl and hyf performed the experiments and wrote the manuscript, and should be considered as first authors. xlc, yy, xwc, tr, wbq helped with the experiment. all authors read and approved the final manuscript. ml contributed to conceive the idea and initiate the project. the authors declare that they have no competing interests. key: cord- -wm c rk authors: evseenko, vasily a; bukin, eugeny k; zaykovskaya, anna v; sharshov, kirill a; ternovoi, vladimir a; ignatyev, george m; shestopalov, alexander m title: experimental infection of h n hpai in balb/c mice date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: wm c rk background: in huge epizooty of h n hpai occurred in russia. it had been clear that territory of russia becoming endemic for h n hpai. in several outbreaks have occurred. to develop new vaccines and antiviral therapies, animal models had to be investigated. we choose highly pathogenic strain for these studies. results: a/duck/tuva/ / belongs to quinghai-like group viruses. molecular markers – cleavage site, k in pb characterize this virus as highly pathogenic. this data was confirmed by direct pathogenic tests: ivpi = . , mld( )= , log eid( ). also molecular analysis showed sensivity of the virus to adamantanes and neuraminidase inhibitors. serological analysis showed wide cross-reactivity of this virus with sera produced to h n hpai viruses isolated earlier in south-east asia. mean time to death of infected animals was , +/- , days. first time acute delayed hemorrhagic syndrome was observed in mice lethal model. hypercytokinemia was determined by elevated sera levels of ifn-gamma, il- , il- . conclusion: assuming all obtained data we can conclude that basic model parameters were characterized and virus a/duck/tuva/ / can be used to evaluate anti-influenza vaccines and therapeutics. influenza a (h n ) virus now becomes a real threat for humans. since , when first human case of h n hpai had been reported, more than people were infected and died [ ] . before attention was attracted to thailand, vietnamese and indonesian viruses. in the beginning of outbreak on quinghai lake occurred [ ] . later "quinghai-like" viruses spreaded to most part of russia, european countries and africa and caused numerous outbreaks. only in russia more than million of different species and sorts of poultry died and been slaughtered [ ] . confirmed cases in azerbaijan, egypt, iraq, and turkey was caused by quinghai-like viruses. earlier hpai viruses were investigated in mice [ , ] and murine models were successively used for reverse genetics made influenza vaccines [ ] . it was shown that h n hpai viruses could have different pathogenicity for mice [ ] . several molecular markers were choused to explain differences. multibasic cleavage site with k in pb designate to highly pathogenic phenotype for mice. also important role of pulmonary cytokines elevation was highlighted [ ] . combination of adaptation for wild waterfowl and high virulence for mammals makes quinghai-like viruses presumably pandemic. also, in future, because of ability for rapid spreading for long distances, this group of viruses can appear in north and south america and cause outbreaks. human disease caused by hpai viruses can be characterized as acute viral pneumonia aggravated by ards, toxic shock and multiple organ failure. system dysfunction mediated by hypercytokinemia and high viral load [ ] . to be ready for new influenza pandemy it is necessary to use animal models, in vaccine and antivirals studies, which most closely reflect human disease. isolates from frsi src vb "vector" repository which were characterized previously were examined for mld , molecular markers of pathogenicity, sensitivity to amantadines and neuraminidase inhibitors, to be candidates for murine model. among the investigated isolates a/duck/tuva/ / has best features to be used. genes of a/duck/tuva/ / were sequenced and analyzed for molecular markers of pathogenicity. also phylogenetic analysis was performed. results are presented in figure . a/duck/tuva/ / belongs to group of qinghailike viruses. ha contains polybasic aminoacids (pqgrrkkkr↓gl) in cleavege site of ha [ ] . the receptor binding domen can be characterized as "avian" [ ] . high pathogenicity to mammals in general correlates with presence of k in pb [ ] . the analysis of non-structural protein (ns ) which also could be contributed for high virulence of h n viruses revealed deletion of amino acids similar to those in h n viruses of genotype z which could be contributed to increased expression of tnf-α and ip- protein in primary human macrophages [ ] . a/duck/tuva/ / con-tained glu in the ns and contained "avian-like" pdzdomain ligand esev [ ] . it was shown that the most recent h n strains isolated in southeast asia were resistant to amantadine and rimantadine; antiviral drugs targeted the m ion channels of influenza a viruses [ , ] . it was also reported about oseltamivir resistant h n viruses isolation from humans [ , ] . to determine the potential sensitivity of studied h n viruses to these antivirals, amino acid sequences of the m and na proteins were analyzed. variants of influenza a viruses resistant to amantadine possessed amino acid substitutions at one of residues ( , , , , and ) in the m protein [ , ] . sequence analysis did not reveal any mutations associated with resistance to amantadine. thus all a/duck/tuva/ / is potentially sensitive to this class of antiviral agents. amino acid residues , , and in the na protein (numbering according to the ha of h subtype) are crucial for the sensitivity of influenza a viruses to neuraminidase inhibitors [ ] ; substitution h →y in the na conferred resistance to oseltamivir was observed in clinical h n isolates [ , ] . sequence comparison of the na protein of a/duck/tuva/ / aligned with the na of n subtype of a/wuhan/ / (h n ) influenza virus showed phenotype potentially sensitive to neuraminidase inhibitors. a/duck/tuva/ / showed wide cross-reactivity with sera against h n hpai viruses isolated earlier in south-eastern asia. hi results can be found in table . these features persuade to use this virus in studies of vaccines made from various h n influenza viruses. first mid and mld for a/duck/tuva/ / were determined (table ) . to determine mean time to death (m.t.d) and c. in several cases ( animals totally) the disease was complicated by severe intestine atony, which can independently lead to death or by pressuring on diaphragm can intensify respiratory failure. we also determined virus titers in several organ tissues. as it was expected the highest titers was observed in lungs - , log eid . brain titers were also high - , log eid . in spleen, liver and kidney tissues virus titers were lower then logeid and considered not significant. we investigated the involvement of several cytokines in immunopathogenesis of experimental h n hpai infection in mice. results of elisa technique revealed alteration of expression both pro-inflammatory and antiinflammatory cytokines after the challenge (figure ). in general, the most marked changes of cytokine levels were observed before the death of mice. the minimal concentration of ifn-γ was detected on day ( . ± . pg/ml), however, its levels enlarged about -fold ( ± pg/ml) during the course of the infection when compared with uninfected animals. on days and systemic production of tnf-α was below the detection limit of the assay. a peak was reached on day by the cytokine ( ± . pg/ml) and its levels remained elevated on day . interestingly, concentrations of il- β in mice after the challenge were significantly lower in comparison with the constitutive expression of the mediator in intact animals. an abrupt decrease of il- β was detected on day post infection, but was followed by step increase from day . after the . -fold enlargement on day the levels of il- decreased dramatically on day , and the highest levels of the cytokine were determined at the end of observation period ( ± pg/ml). the constitutive production of il- was undetectable. the dynamics of il- showed a gradual growth with the maximum level ( . ± . pg/ml) reached before the death of mice. we observed statistically significant increase of il- after the challenge. concentrations of the cytokine retained constant in infected mice, except the unexpected decline occurred on day . the expression of il- could not be detected throughout the entire period of observation. until avian influenza was regional problem of sev- [ ] and question "why had only some wild waterfowl died?" is still unclear. most of the outbreaks in russia associated with wild birds. the same time viruses adapted to wild birds are extremely pathogenic for poultry and mice. this "competitive advantage" makes quinghailike viruses most probable candidate to be precursor for new pandemic influenza virus. at the same time pathogenesis of different (phylogenetical clades) hpai reveal common causes. the principal causes of rapid mice death after infecting with hpai are primary viral pneumonia, ards, lesions of central nervous system and multiple organ failure. our data suggest that a/duck/tuva/ / strain of hpai caused lethal pneumonia and spread systemically to the brain in balb/c mice. lesion of respiratory epithelium and following an activation of monocytes/macrophages results in a release of proinflammatory cytokines (tnf-α, il- ) which are a hallmark of ards in murine model [ ] . despite powerful anti-influenza virus effects of tnf-α in lung tissue, as it was described previously [ ] , we consider that elevated production of the cytokines seems to be crucial in the pathogenesis of hpai infection. moreover, it was shown that lethal h n viruses are resistant to antiviral effects of interferons and tnf-α [ ] . virus-induced overexpression of tnf-α as well as high ifn-γ lead to activation of endothelium and imbalance in blood coagulation system [ ] . this may explain the hemorrhagic syndrome as observed in some of animals. to pay attention that il- is a potent inducer of ifn-γ synthesis by blood mononuclear cells [ ] , we concluded the same cytokines hyperproduction reflects macrophage overactivation and subsequent hypercytokinemia. this cascade of events phylogenetic tree based on full length sequencesof ha figure phylogenetic tree based on full length sequencesof ha. nucleotide sequences were analyzed by using the neighborjoining method with bootstraps. the phylogenetic tree was rooted to the ha gene of a/goose/guangdong/ / (h n ) virus. including inflammatory mediator production, changes in blood coagulation system and microvascular permeability was denoted as systemic inflammatory response syndrome (sirs) [ ] . on the other hand, we proposed that the prominent production of il- from the early stages of the experimental hpai infection was the compensatory response to overproduction of proinflammatory cytokines such as tnf-α, il- and il- . however, the role of il- , which principle function seems to be containment and eventual termination of inflammation [ ] , in hpai pathogenesis is unclear. also there is an uncertain discrepancy between undetectable expression of il- and high levels of other th -cytokines (ifn-γ and il- ). summing up, in our study balb/c mice infected with hpai, strain a/duck/tuva/ / , appeared to be able to produce the innate immune response, which culminated to the development of shock and subsequent multiple organ failure. the main characteristics of our model are comparable to the previously described fatal cases of h n influenza in humans [ , ] . proposed model reflects lesions not only same organs but also mediating levels of some (ifn-γ, il- , il- ) cytokines in terminal conditions. the implication of different cytokines in immunopathogenesis of experimental hpai is beyond question. but to understand exact mechanisms, which determine the disease outcome, further experiments remain to be done. all experiments were performed in bsl + facilities of fsri src vb "vector" of rospotrebnadzor licensed for working with highly pathogenic avian influenza viruses. stock of a/duck/tuva/ / was produced in days-old chicken embryos. allantoic fluid was aliquoted and stored at - °c. the infectivity of stock viruses was determined in days-old embryonated chicken eggs; titers were calculated by the method of reed and muench [ ] and were expressed as log of % egg infective dose (eid ) in ml of allantoic fluid. viral rna was isolated from virus-containing allantoic fluid with the rneasy mini kit (qiagen, valencia, ca) as specified by the manufacturer. uni- primer was used for reverse transcription. pcr was performed with a set of primers specific for each gene segment of influenza a virus [ ] . pcr products were purified with the qiaquick pcr purification (qiagen). sequencing was done with beckman coulter genom-elab™ methods development kit dye terminator cycle sequencing according instructions of manufacturer. primers for sequence were obtained from e. hoffman (sjcrh, memphis, tn). sequence products were analyzed on automatic sequence analyzer beckman coulter ceq . phylogenetical analysis was done on ha full gene sequence dq using mega . software. phylogenetical tree was built by neighbor-joining method; matrix of distances was counted with p-distance algorithm. reliability of clades was checked with bootstrap analysis with replications. other genes in genbank dq -dq . cross-reaction of a/duck/tuva/ / was defined by hemagglutination inhibition test (hi) with . % crbc [ ] with a panel of antisera against h n hpai. six-week-old inbred male balb/c mice (vivarium of frsi src vb "vector"). animals were placed to individual cages with food and water available ad libitum. to determine the mld and mid , mice were anaesthetized by diethyl ether inhalation and infected intranasally with µl -fold serial dilutions of allantoic fluidin pbs (ph , ). each group contained animals. animals were observed daily for days for mortality (mld ) or sacrificed on day after the challenge with following virus detection in the lungs by inoculation of days-old embryonated chicken eggs (mid ). mld and mid were calculated by the method of reed and muench. animals from group where mld had been observed were taken to determine virus titers in lung, spleen, kidneys, and liver and brain tissues. mind time to death (m.t.d) was calculated as previously described [ ] . pathogenicity to chickens was determined by ivpi test [ ] . all animal studies were performed according protocols approved by animal care & use committee of fsri src vb "vector". to determine ifn-γ, tnf-α, il- , il- , il -β, il- we use elisa r& d systems kits (minneapolis, mn, usa). serum levels of il- were measured using commercial mouse il- elisa test kit (mbl, nagoya, japan). detection limits were as follows: tnf-α, less then , pg/ml; il -β, , pg/ml; il , , pg/ml; il , , pg/ml; il- , pg/ml. sera was taken on , , , , days and aliquots and stored - °c upon usage. day was chosen because m.t.d defined earlier in the work was , ± , days. statistics was performed with student t-test. values p < , considered to be reliable. world health organization: epidemic and pandemic alert and response (epr) highlypathogenic h n influenza virus infection in migratory birds avian influenza in siberia - : laboratory and epidemiological studies, antiepidemic measures during the epizooty of avian influenza in poultry in siberian and ural federal regions of the russian federation neurovirulence in mice of h n influenza virus genotypes isolated from hong kong poultry in avian influenza (h n ) viruses isolated from humans in asia in exhibit increased virulence in mammals efficacy of h influenza vaccines produced by reverse genetics in a lethal mouse model avian influenza (h n ) viruses isolated from humans in asia in exhibit increased virulence in mammals pathogenesis of hong kong h n influenza virus ns gene reassortants in mice: the role of cytokines and b-and t-cell responses fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia a simple method for estimating fifty percent endpoints universal primer set for the full-length amplification of all influenza a viruses us department of health, education and welfare immunology series statistical methods in microbiological studies a color atlas and text on avian influenza. papi editore influenza: emergence and control structure and receptor specificity of the hemagglutinin from an h n influenza virus the polymerase complex genes contribute to the high virulence of the human h n influenza virus isolate a/ vietnam/ / h n influenza: a protean pandemic threat large-scale sequence analysis of avian influenza isolates genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia detection of amantadine-resistant variants among avian influenza viruses isolated in north america and asia avian flu: isolation of drug-resistant h n virus oseltamivir resistance during treatment of influenza a (h n ) infection themolecular basis of the specific anti-influenza action of amantadine influenza virus m protein has ion channel activity molecular mechanisms of influenza virus resistance to neuraminidase inhibitors acute respiratory distress syndrome induced by avian influenza a (h n ) virus in mice tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells lethal h n influenza viruses escape host anti-viral cytokine responses the interactions between inflammation and coagulation regulation of human cytolytic lymphocyte responses by interleukin- toward an epidemiology and natural history of sirs (systemic inflammatory response syndrome) interleukin- and the interleukin- receptor this work was supported by bio industry initiative (bii) of the us department of state grant istc # . the author(s) declare that they have no competing interests. ve carried out molecular genetic analysis, performed animal studies, design of experiments and drafted manuscript. eb performed immunoassays and obtained data analysis. az participated in animal studies. ks assisted in animal studies. vt was responsible for sequence. gi participated in study design and coordination. as carried out coordination. all authors read and approved the final manuscript. key: cord- - b ljjq authors: lon, jerome rumdon; bai, yunmeng; zhong, bingxu; cai, fuqiang; du, hongli title: prediction and evolution of b cell epitopes of surface protein in sars-cov- date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: b ljjq background: in order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. the polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. the discovery of epitopes is helpful to the development of sars-cov- vaccine. methods: the analyses were related to epitopes on proteins, including spike (s), envelope (e) and membrane (m) proteins, which are located on the lipid envelope of the sars-cov- . based on the ncbi reference sequence: nc_ . , the conformational and linear b cell epitopes of the surface protein were predicted separately by various prediction methods. furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of sars-cov- were obtained from the gisaid. results: epitopes were predicted, including linear epitopes and conformational epitope. one of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. it is worth mentioning that all identified epitopes were conserved in nearly sars-cov- genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. conclusion: the findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus. in late december , a novel coronavirus was officially named as sars-cov- by the international committee on taxonomy of viruses (ictv) and identified as the pathogen causing outbreaks of sars-like and mers-like illness in chinese city of wuhan, which was a zoonotic disease. as of august , , the outbreak of sars-cov- has been reported in many areas of the world, with more than , , people infected [ ] . with an alarming epidemicity, the reproductive number of sars-cov- has been computed to around . [ ] . according to the data in the national genomics data center (ngdc, https ://bigd.big.ac.cn/ncov/), , genomic variations of sars-cov- has been reported at : (gmt + ) on august , , which has aroused widespread concern. the b cell epitope of viral surface protein can specifically bind to the host's b cell antigen receptor and induce the body to produce protective antibody and humoral immune response. the discovery of epitopes is helpful to the development of sars-cov- vaccine and the understanding of sars-cov- ′s pathogenesis [ ] . proteins embedded in the virus envelope of sars-cov- have been identified, including spike (s), envelope (e) and membrane (m) proteins. at present, due to the lack of study of the crystal structure of surface protein of sars-cov- , the study of epitopes, is time-consuming, power-consuming, costly and difficult [ ] , especially the conformational epitopes that depend on accurate protein structures. in this work, we analyzed the surface protein (s, e and m protein) of sars-cov- and predicted the structures with bioinformatics methods. on this basis, we predicted the linear and conformational b cell epitopes, analyzed the conservation of the epitopes, the adaptability and other evolutionary characteristics of the surface protein, which provided a theoretical basis for the vaccine development and prevention of sars-cov- . however, the results still need some experimental confirmation to ensure the validity of the application. all of the analyses and prediction were based on the ncbi reference sequence: nc_ . . on the basis of previous research of our group, genome sequences from gisaid (up to april th, ) were downloaded to construct a dataset for conservation analysis (additional file : table s ) [ ] . the structure of s protein(pdb id: × p) was downloaded from rcsb pdb [ ] , which has a resolution of . Å [ ] .the data sets for s, e, and m protein were obtained by extracting the corresponding locations of the reference genome. the physical and chemical properties of target protein were analyzed by the port-param tool in expasy(expert protein analysis system) [ ] , an online practical analysis kit for proteomics, including the primary structure of the target protein, molecular formula, theoretical isoelectric point, the protein instability index(the index < means the protein was stable) and the location information. online software, protscale, was used to deeply analyze the hydrophilicity and hydrophobicity of target protein and the distribution of hydrophilicity and hydrophobicity of polypeptide chains [ ] . sars-cov- carried the s/e/m proteins through the virus envelope, the transmembrane region of the protein was predicted online by tmhmm . [ ] . with the amino acid sequences of the surface protein of sars-cov- of nc_ . as templates, we predicted the d structure of e and m protein through the online server swiss-model [ ] based on homology modeling method, selected the optimal structure based on the template identity and gmqe value [ ] , and the rationality of the structure was evaluated by ramachandran plot [ ] with pdbsum server. the structures were displayed and analyzed by swiss-pdb viewer v . [ ] . based on the structures, the conformational b cell epitopes were predicted by seppa . [ ] and ellipro [ ] respectively, and the conformational b cell epitopes, which were predicted by all of the two methods were selected for the further analysis. the protean module of dnastar was used to predict the flexibility [ ] , surface probability [ ] and antigenic index [ ] of the target protein of sars-cov- . the linear b cell epitope was predicted by abcpred [ ] and bepipred . [ ] respectively and the common predicted linear b cell epitopes from two methods were selected for the further analysis. coupled with the secondary structure, the tertiary structure and the glycosylation sites [ ] , the linear b cell epitopes were finally determined. based on the pdb model and the multiple alignment result, we used the consurf server to analyze the conservation of amino acid sites of the epitopes online [ ] . the conservation of epitopes on the surface protein of sars-cov- was analyzed by multiple alignment with mafft and logo was drawn with weblogo [ , ] . the primary structure and physicochemical properties of the s/e/m protein were analyzed. the results revealed that the s, e and m protein have average hydrophilic indexes of − . , . and . , respectively. on the basis of hydrophilicity, the s and m protein showed amphipathic properties, the e protein showed hydrophobic (additional file : figure s ). according to the prediction, there was an outside-in transmembrane helix in residues from position th to position th at the n-terminal of the s protein, which was almost consistent with the study indicating that the transmembrane domain of s protein was at the position from to th [ ] , an inside-out transmembrane helix in residues from position th to position th at the n-terminal of the e protein. two outside-in transmembrane helices of the m protein, one was in residues from position th to position th, the another one was in residues from position th to position th, and an inside-out transmembrane helix of the m protein in residues from position st to position rd at the n-terminal, was predicted (additional file : figure s ). each transmembrane region corresponds to a hydrophobic peak in the hydrophilic index curve. the protein instability index of the s, e and m protein were . . and . , which revealed that all of the s, e and m protein was stable. the optimal template for homology modeling of the e protein of sars-cov- was the e protein of sars (pdb id: × . ), with the sequence identity of . % and the gmqe score of . . according to the evaluation of the structure by ramachandran plot (fig. a) , % of the residues were located in the most allowed regions ( table ), indicating that the structure was reliable. the e protein of sars-cov- is a pentamer (fig. b) , which can be divided into the concentrated transmembrane part and the head located outside the envelope. the head is mainly composed of α-helix, irregular curl and turn, which is exposed to the envelope and contributes to the formation of epitopes. the tail is mainly composed of long α-helix, most of which are embedded in the envelope, hindering the formation of epitopes. the optimal template for homology modeling of the m protein of sars-cov- was the effector protein zt-kp - (pdb id: qpk. . a), with the sequence identity of . % and the gmqe score of . . the sequence identity between the optimal template and the m protein of sars-cov- and the gmqe score are too low, so that the template is not suitable for homology modeling. all linear b cell epitopes of the surface protein were filtered according to the following criteria: ( ) region with high surface probability (≥ . ), strong antigenicity(≥ ) and high flexibility; ( ) excluding the region with α-helix, β-sheet and glycosylation site (fig. ) ; ( ) in line with the prediction by bepipred . (cut off to . ) and abcpred (cut off to . ). based on the results obtained with these methods and artificial optimization, we removed epitopes that are too long to be suitable for applicate, potential linear b cell epitopes of the s protein were predicted ( table , fig. a ), including - aa, - aa, - aa, - aa, and they were named as the epitope a, b, c, d, respectively; epitope of the e protein was selected( - aa) and named as the epitope e ( table , fig. c ); epitope of the m protein was selected ( - aa) and named as the epitope g ( table ). the d structure prediction and ramachandran plot analysis of the e protein. a the ramachandran plot analysis of the d structure of the e protein (without gly and pro). all of the residues located on the allowed region. indicating that the structure was reliable from a thermodynamic point of view. b the d structure of the e protein predicted by homology modeling. it is a pentamer with ion channel activity [ ] . its head is short, the middle of the tail is a transmembrane region which help the e protein embed in the envelope of sars-cov- residues in disallowed regions . with the structure of s protein (pdb id: × p), the conformational b cell epitopes of surface protein were predicted with ellipro and seppa . with the default threshold of . and . , respectively. one conformational b-cell epitope ( - aa) of e protein was predicted (table ) , which is consistent with the linear epitope e. similarly, this region located on the outside (fig. c) , and we selected it as a dominant conformational epitope and named f. however, the conformational epitope of the m protein could not be predicted due to the failure of credible homology modeling. the consurf server was used to predict epitope conservative sites with the structure of surface proteins and the alignment results in our dataset. due to the lack of crystal structure of m protein, the epitope g only applies interestingly, the high antigenicity peaks of all three proteins were in the region where the α-helix is relatively sparse, which may be related to the fact that the α-helix structure of the helix prevents continuous residues from being located on the surface table the composition and the antigenic index of the epitopes of sars-cov- the scores of the epitope e and the epitope g were calculated by ellipro, the others were calculated by bepipred . . the epitopes a, b, c and d belong to s protein, the epitopes e and f belong to e protein and they are coincident, the epitope g belongs to m protein data sets to calculate conservation. in the dataset, the epitopes of s, e and m protein were basically conservative (table , additional file : figure s ). further calculation of the conservatism of the epitopes in the dataset was carried out, and the average score of all the epitopes was less than , which could be considered as conservative epitopes. epitope e and epitope f from e protein had the lowest scores and showed the highest conservatism. however, it is worth noting that this value is an overall assessment of the epitopes. residues no. , of epitope d and of epitope g, as a single residue, showed a conservative score greater than respectively, which revealed a risk of mutation. sars-cov- caused huge impact to human production, living and even life, and has become a major challenge confronting the whole world. development of vaccine is one of the effective means of long-term prevention of the virus. epitope vaccine is the trend of development of vaccine due to the advantages of strong pertinence, less toxic and side effects and easy to transportation and storage [ ] . a group founded in march by preston estep, calling themselves "the rapid vaccine partnership" (radvac), has developed a very simple vaccine. in early july, radvac published a white book detailing the vaccine they developed (https ://radva c.org/). the radvac vaccine is a "subunit" vaccine because it is composed of fragments of a pathogen, in this case it was peptide, which is essentially a short fragment of a protein that matches the sars-cov- section but does not cause disease. subunit vaccines are already used for diseases such as hepatitis b and human papillomavirus, and a number of companies are developing subunits for covid- , including novavax biotechnology. reliable epitopes are particularly important for the development of subunit vaccines. (https :// www.techn ology revie w.com/ / / / /georg e-churc h-diy-coron aviru s-vacci ne/). the determination of epitopes is the basis of the development and application of vaccine, and the clinical diagnosis.herrera et al. [ ] reported antigenic analysis of s protein obtained by elisa, but did not study the epitopes. the conserved epitopes were predicted based on the calculation by us, which provided more reference for the immunological study of s protein. vashi et al. predicted some epitopes based on the structure of s protein [ ] . although their studies predicted both b-cell and t-cell epitopes of s protein, they did not discuss the conservation of epitopes. we effectively supplement the study of epitopes with the conservative analysis based on a large amount of data, which can ensure the long-term effect and stability of epitopes in the application process. at the same time, their study is limited to s protein, while our study on e and m protein provides more options. moreover, walls et al. [ ] reported the use of conservative glycosylation sequence in s protein of sars-cov can stimulate neutralizing antibody against sars-cov- , the epitope g is the linear epitope and the f is the conformational epitope, which are coincide and the study of the yuan et al. [ ] reported that they researched the recognition of epitopes and antibodies by parsing the structure of antibody cr from rehabilitation in patients with sars. wang et al. [ ] reported a kind of human monoclonal antibodies, which could neutralize the sars-cov- , from the cell culture. what these studies have in common is that they are based on some immune responses that have already occurred. in contrast, our calculation in the computer environment is faster, but the accuracy still needs to be verified experimentally. the two methods form an effective complement. currently, the methods which were mainly used are x-ray scattering method, immune experiment method and bioinformatics method. the first two are time-consuming and laborious, while the bioinformatics method is gaining more and more credibility among researchers [ , , ] . there are many factors to be considered in the prediction of epitopes by bioinformatics method, such as the surface probablity and flexibility of the epitopes. at the same time, it is necessary to exclude the structurally stable and non-deformable α-helix, β-sheet, glycosylation sites which may obscure the epitopes or alter the antigenicity, etc. [ ] . even so, the predicted epitopes are still inaccurate [ ] . our work takes the intersection of above methods to predict, which greatly improves the stability of the prediction. compared with the current study on sars-cov- , this work adopted various prediction methods and d structure databases developed in recent years, which were based on artificial neural network, hidden markov model (hmm), support vector machine(svm), etc., such as abcpred, bepipred . , seppa . , iedb, etc. compared with prediction by a single method [ ] , on the basis of a single protein [ ] or on the basis of epitopes of sars [ ] , these methods and databases greatly improved the accuracy of prediction and had more bioinformatic meaning. we comprehensively analyzed the prediction results from the tools which were widely used, set up screening criteria on the basis of primary structure, secondary structure and tertiary structure, so that the prediction results would more accurate and reliable. the s protein, the e protein and the m protein are surface proteins of sars-cov- that form the outer table the the calculation was independent and based on the sars-cov- data set layer of the coronavirus and protect the internal rna, which have the potential as antigenic molecules. however, considering the current study on the epitopes prediction of sars-cov- [ ] and due to the fact that s protein has been reported to be the directly binding molecule of sars-cov- to ace [ ] , the prediction of epitopes is mainly focusing on the s protein, with few studies on the e protein and the m protein. in this work, we analyzed the s protein, the e protein and the m protein and predicted their epitopes. on this basis, b cell epitopes were predicted, including conformational and linear b cell epitopes, one of the conformational and one of the linear are coincide. all of the epitope a, b, c, d located on the surface of the tail of the s protein, which is relatively easy to bind. the epitope e and the epitope f located at the end of the head of the e protein coincide, and this may be explained by the fact that they are all consecutive and the secondary structure avoiding the α-helix and the β-sheet. the epitope g is derived from the m protein, and the structure and conservation could not be determined due to the inability to predict reliable structure. however, it could be inferred from the surface probability scores that the epitope g is more likely to be located on the surface of the m protein. the higher the conservation score calculated by the consurf server is, the more likely the site is to be mutated in the evolutionary process. when the score < , the site is likely to be a conservative site; when the score is between and , the site is a site which is likely to be a relatively easy mutation; when the score > , the site is likely to be an easy mutation site [ ] . in the epitopes obtained, all the epitopes of the s, e, m protein were absolute conservative among all sars-cov- sequences. the conservation of the epitope g could not be calculated by the pdb file. our work provides identified and conserved sites for further study. mutations that occur during the spread of the virus can cause significant resistance to vaccine development. for example, the recently reported mutation of amino acid of s protein [ ] not only affects the ability of the virus to transmit, but also may affect the efficacy of vaccines involving this site. our work provides reliable candidates for the development of epitope vaccines, but the application value of the epitopes needed further experimental verification. for example, the antigenicity of the epitope could be tested. although the epitopes could be integrally considered to be conservative, the independent residues of these epitopes could still easy to mutate. epitopes d and e had two and one residues, respectively, with conservative scores greater than , meaning that they were at risk for a single point mutation. more attention should be paid to these two epitopes in application. the epitope detection in glycoproteins is significant to the study of the immunoreaction of sars-cov- , but its challenge is less reliable than the epitope detection due to the presence of glycan [ ] . in addition, sars-cov- would mutate frequently, and the epitopes predicted might mutate too, so conservative epitopes analyzed in the present study might be more reliable. according to the data from ngdc, the variation frequencies of s, e, and m proteins were . , . , and . , respectively. under the condition of relatively high variation frequency, the conservation of the proteins was analyzed to identify the epitopes with low mutation risk, which were important for the development of long-term and stable vaccines. however, this work is limited. without the molecular dynamic analysis, the binding between epitopes and antibodies was not simulated to further determine the availability of epitopes, but researches from different perspectives can provide more epitopes choices for subsequent studies. in this work, we predicted reliable epitopes: a, b, c, d, e/f and g. the reliability of the epitopes of the s protein was relatively better than that of the epitopes of the e protein and the m protein, indicating that the s protein is still the optimal choice for the prediction of epitopes and the development of vaccine. all of the epitopes were able to achieve high conservation in sars-cov- , therefore, the epitopes not only have the potential to be directly applied on the treatment in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus. in addition, although various factors of prediction were integrated in this work, more experimental data are needed to further verify whether all the epitopes can induce the body to produce corresponding antibodies and generate specific humoral immunity, due to the limited data set and other factors. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . additional file . figure s : deep analysis of hydrophilicity and hydrophobicity of surface protein of sars-cov- . the online software, protscale, was used to predict the hydrophilicity and hydrophobicity of the surface protein deeply. a. the s protein has a maximum score of hydrophobicity, . at the th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, - . at the th site, which revealed a strong hydrophilicity. the score of hydrophilicity and hydrophobicity on the polypeptide chain of s protein constantly fluctuates, with most of the scores being negative, which revealed the possibility that the protein had bisexual properties on the basis of hydrophilicity. b. the e protein has a maximum score of hydrophobicity, . at the st and the th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, - . at the th site, which revealed a strong hydrophilicity. most of the scores of the residues being positive, which revealed the possibility that the protein has obvious hydrophobicity. c. the m protein has a maximum score of hydrophobicity, . at the th site, which revealed a strong hydrophobicity; a minimum score of hydrophobicity, - . at the th and the th site, which revealed a strong hydrophilicity. the scores of hydrophilicity and hydrophobicity on the polypeptide chain of m protein showed large fluctuations, and the number of positive scores and negative scores were similar, the positive scores accounted for the majority, which revealed the possibility that the protein had bisexual properties on the basis of hydrophobicity. additional file . figure s : the transmembrane region of the surface protein of sars-cov- . the s, e and m protein are embedded in the envelope of sars-cov- , the transmembrane helix was predicted by tmhmm . server. all of three amino acid indexes were higher than , indicating the reliability of the prediction. a. for the s protein, an outsidein transmembrane helix was predicted in the residues of amino acids from position th to position th at the n-terminal. the amino acid index was . . b. for the e protein, an inside-out transmembrane helix was predicted in the residues of amino acids from position th to position th at the n-terminal. the amino acid index was . . c. for the m protein, outside-in transmembrane helices were predicted, which were a helix in the residues of amino acids from position th to position th and a helix in the residues of amino acids from position th to position th at the n-terminal. an inside-out helix was predicted in the residues of amino acids from position st to position rd at the n-terminal. the amino acid index was . . the calculation of the transmembrane pattern and data has clarified the position and direction of the protein in the virus, which is of great significance for the understanding of the availability of the antigen when predicting the epitopes, the epitopes located outside the virus has significant application advantages. additional file . figure s : the antigenic conservation of the surface protein in sars-cov- . the overall height of each stack is proportional to the sequence conservation, measured in bits, at that position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position. all the epitopes in the data set are highly conservative, and the serial numbers (a-g) in the figure represent the epitopes a-g respectively. additional file . table s : the list of the id of genomes in dataset. coronavirus -ncov: a brief perspective from the front line the reproductive number of covid- is higher compared to sars coronavirus recent advances in b-cell epitope prediction methods bioinformatics resources and tools for conformational b-cell epitope prediction comprehensive evolution and molecular characteristics of a large number of sars-cov- genomes reveal its epidemic trends rcsb protein data bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy characterization of the sars-cov- s protein: biophysical, biochemical, structural, and antigenic analysis the proteomics protocols handbook sequence and structure-based prediction of eukaryotic protein phosphorylation sites swiss-model: homology modelling of protein structures and complexes procheck: a program to check the stereochemical quality of protein structures swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling seppa . -enhanced spatial epitope prediction enabling glycoprotein antigens ellipro: a new structure-based tool for the prediction of antibody epitopes prediction of chain flexibility in proteins induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide the antigenic index: a novel algorithm for predicting antigenic determinants convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc, research is always in progress. learn more biomedcentral.com/submissions ready to submit your research ready to submit your research ? choose bmc prediction of continuous b-cell epitopes in an antigen using recurrent neural network improved method for predicting linear b-cell epitopes prediction of n-glycosylation sites in human proteins consurf : an improved methodology to estimate and visualize evolutionary conservation in macromolecules weblogo: a sequence logo generator sequence logos: a new way to display consensus sequences severe acute respiratory syndrome coronavirus- (sars-cov- ), a newly emerged pathogen: an overview immuno-informatics: mining genomes for vaccine components understanding the b and t cell epitopes of spike protein of severe acute respiratory syndrome coronavirus- : a computational way to predict the immunogens structure, function, and antigenicity of the sars-cov- spike glycoprotein a highly conserved cryptic epitope in the receptor binding domains of sars-cov- and sars-cov a human monoclonal antibody blocking sars-cov- infection an introduction to epitope prediction methods and software the evolutionary pattern of glycosylation sites in influenza virus (h n ) hemagglutinin and neuraminidase a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of -ncov cross-reactive antibody response between sars-cov- and sars-cov infections potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody mutation feature analysis on epitope and receptor binding sites of influenza a h n hemagglutinin spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov- analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the student entrepreneurship and innovation center of the school of biology and biological engineering, south china university of technology, also provided a lot of help during the preparation of the project. authors' contributions jl conceived the study and participated in its design and coordination. hd participated in the design of the study and helped draft the manuscript. yb participated in analysis of conservation, sequence alignment and manuscript drafting. bz participated in antigenic prediction. fc participated in drafting the manuscript. all authors read and approved the final manuscript. the viral genomes described in detail here were deposited in ncbi, genbank and gisaid. not applicable. not applicable. the authors declare that they have no competing interests.received: may accepted: october key: cord- - w l a authors: garry, courtney e; garry, robert f title: proteomics computational analyses suggest that the bornavirus glycoprotein is a class iii viral fusion protein (γ penetrene) date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: w l a background: borna disease virus (bdv) is the type member of the bornaviridae, a family of viruses that induce often fatal neurological diseases in horses, sheep and other animals, and have been proposed to have roles in certain psychiatric diseases of humans. the bdv glycoprotein (g) is an extensively glycosylated protein that migrates with an apparent molecular mass of , to , kilodaltons (kda). bdv g is post-translationally cleaved by the cellular subtilisin-like protease furin into two subunits, a kda amino terminal protein gp and a kda carboxyl terminal protein gp . results: class iii viral fusion proteins (vfp) encoded by members of the rhabdoviridae, herpesviridae and baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. proteomics computational analyses suggest that the structural/functional motifs that characterize class iii vfp are located collinearly in bdv g. structural models were established for bdv g based on the post-fusion structure of a prototypic class iii vfp, vesicular stomatitis virus glycoprotein (vsv g). conclusion: these results suggest that g encoded by members of the bornavirdae are class iii vfps (gamma-penetrenes). members of the bornaviridae are enveloped with nonsegmented negative-stranded rna genomes. the type member is borna disease virus (bdv), the causative agent of borna disease, an often fatal neurological disease occurring mainly in horses and sheep in endemic regions of central europe. the natural host range, prevalence, and geographic distribution of bdv are broad [ ] . for example, recent studies demonstrated the existence of an avian reservoir of diverse bornaviruses [ ] and provided evidence that bornaviruses are the etiologic agent for proventricular dilatation disease, a neuroinflammatory disease of psittacine birds [ ] . it is yet to be established whether bornaviruses causes any overt disease in humans. however, correlative evidence exists linking bdv infection with neuropsychiatric disorders, such as bipolar disorder [ , ] . bdv is noncytolytic and highly neurotropic [ ] . although viral rna and proteins are readily detectable in bdvinfected cells, production of cell-free virions or cell-associated infectivity is absent or extremely low [ ] . bdv propagates via cell-to-cell spread within the central nervous system and cultured cells in the absence of detectable assembled virions [ , ] . it is likely that bdv initially inter-acts with the plasma membrane, followed by endocytosis and penetration by membrane fusion; cell-to-cell spread of bornaviruses may involve distinct mechanisms [ ] . the bdv genome contains at least six open reading frames (orf), and the product of bdv orf iv corresponds to the type i surface glycoproteins found in other viruses of the order mononegavirales. the bdv glycoprotein (g, gp-c, gp ) migrates due to extensive glycosylation with an apparent molecular mass of , to , kilodaltons (kda). bdv g is post-translationally cleaved by the cellular subtilisin-like protease furin [ , ] . the amino terminal product (gp , gn, gp ) has an apparent molecular mass of kda. gp was initially difficult to detect in infected cells or virions due to its high glycan content, which shields antigenic sites from antibodies. however, a peptide-generated antibody was used to demonstrate the presence of gp in virions and infected cells [ ] . the carboxyl terminal product, molecular mass kda (gp , gc, gp ), is less heavily glycosylated and readily detected in infected cells and virions. the uncleaved product g accumulates in the endoplasmic reticulum and perinuclear region. g is not detected on the plasma membrane, whereas gp , but not gp , accumulates at the cell surface [ ] . there are conflicting results as to the presence of uncleaved g in virions [ , ] . gp contains several hydrophobic sequences, and appears capable of mediating cell:cell fusion in the absence of other bdv surface proteins [ , ] . gp linked to the vesicular stomatitis virus glycoprotein transmembrane domain (vsv g tm) domain is sufficient, in the absence of gp , for receptor recognition, cell fusion and entry [ ] . the structures of the bornavirus glycoproteins have not been determined, and it is unknown whether or not these proteins belong to any of the three known classes of proteins that mediate membrane fusion and entry of enveloped viruses. orthomyxoviruses (excepting members of the thogotovirus genus, which are insect pathogens), retroviruses, paramyxoviruses, filoviruses, arenaviruses, and coronaviruses encode class i viral fusion proteins (vfp, aka class i or α-penetrenes). class i vfp contain a "fusion peptide," a cluster of hydrophobic and aromatic amino acids located at or near the amino terminus that initially interacts with the target cell membrane (plasma membrane or vesicle membrane) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most class i vfp have an aromatic amino acid (aa) rich pre-membrane domain, while all have a carboxyl terminal anchor. the post-fusion forms of class i viral fusion proteins have an extended amino terminal helix (n-helix, hr ), and a carboxyl terminal helix (c-helix, hr ) or "leash domain" that mediate trimerization. members of the flavivirus genus of the flaviviridae and the alphavirus genus of the togaviridae encode class ii viral fusion proteins (class ii or β-penetrenes) possessing three domains (i-iii) of mostly antiparallel β-sheets, a membrane proximal α-helical stem domain and a carboxyl terminal anchor [ ] [ ] [ ] . the fusion loops of class ii vfp are internal and located in domain ii, the fusion domain. hepaciviruses and pestiviruses, the two other genuses in the flaviviridae, appear to encode truncated class ii vfp [ ] . proteomics computational analyses and other studies suggest that the carboxyl terminal glycoproteins (gc) of bunyaviruses, tenuiviruses and caenorhabditis elegans retroviruses, are class ii vfp [ , ] . the third class of vfp (class iii or γ-penetrenes) is represented by glycoprotein (g) of rhabdoviruses and glycoprotein b (gb) of herpesviruses. class iii vfp contain a fusion domain, which is similar structurally to the fusion domains of class ii vfp, other β-sheet domains, an extended α-helical domain in the post-fusion form, a membrane proximal stem and a carboxyl terminal anchor [ ] [ ] [ ] . the extended α-helices in the post-fusion forms of rhabdovirus g and herpesvirus gb are involved in trimerization, a similar function served by the long α-helices in the post-fusion structures of class i vfp. our prior proteomics computational analyses suggested that the fusion proteins of group i nucleopolyhedroviruses (npv) of the baculoviridae and members of the thogotovirus genus of the orthomyxoviridae, which together form the gp superfamily, are also class iii vfp [ ] . a recent xray crystallographic study, published after our prior manuscript, confirmed that gp superfamily members are class iii vfp [ ] . here, proteomics computational analyses are presented suggesting that bornavirus g are class iii vfp. sequence and structural comparisons were performed for borna disease virus strain v glycoprotein isolated from a horse (accession number np ). additional bornavirus g sequences used in the analyses included abh , cac (horse), aal (sheep), abh (rabbit), acg (avian -aratinga solstitialis), and aaa (human). we also made comparisons of bornavirus g with thogato virus strain siar envelope glycoprotein precursor (p ), the autographa california multiple nucleopolyhedrovirus gp superfamily protein (p ) and other gp superfamily members. representatives of g from six genera of the rhabdoviridae were also used for sequence and structural comparisons: vesiculovirus: vsv strain indiana (aaa ); lyssavirus: rabiesvirus strain street (aaa ); ephemerovirus: bovine ephemeral fever virus structural g (p ) and nonstructural g(p ); novirhabdovirus: infectious hematopoietic necrosis virus (caa ); cytorhabdovirus: lettuce necrosis yellows virus glycoprotein (lyp ); nucleorhabdovirus: rice yellow stunt virus (ab ) and an unclassified rhabdovirus: taastrup virus (ay ). we also compared bornavirus g to vfp of representative members of the herpesviridae, flaviviridae, togaviridae, and bunyaviridae. methods developed by william gallaher and coworkers to derive models of vfp have been described previously [ , , , ] . william pearson's lalign program http:/ /www.ch.embnet.org/software/lalign_form.html, which implements a linear-space local similarity algorithm [ ] was used to perform regional alignments. phd (columbia university bioinformatics center, http:// cubic.bioc.columbia.edu/predictprotein/), which is part of the proteinpredict suite was the preferred method of secondary structure prediction [ ] . domains with significant propensity to form transmembrane helices were identified with tmpred (expasy, swiss institute of bioinformatics, http://www.ch.embnet.org/software/ tmpred_form.html). tmpred is based on a statistical analysis of tmbase, a database of naturally occurring transmembrane glycoproteins [ ] . sequences with propensity to interface with a lipid bilayer were identified with membrane protein explorer version . from the stephen white laboratory using default settings [ ] , which can be used to calculate scores on the wimley-white interfacial hydrophobicity scale (wwihs) [ ] . proteomics computational tools have been applied successfully to discover potential structures of the vfp of retroviruses, filoviruses, coronaviruses, and baculoviruses [ , , , , , , , ] . subsequent studies, using xray crystallography and other methods, confirmed all essential features of our structural models suggesting that these modeling methods are highly robust [ , , ] . the phd algorithm predicts protein secondary structure from multiple sequence alignments by a system of neural networks, and is rated at an expected average accuracy of % for three states, helix, strand and loop [ ] . this algorithm predicts that there is a region that forms an extended α-helix in bvd g (aa - ) and other bornaviruses (not shown). a prominent feature of class iii vfps is an extended α-helix beginning near the carboxyl terminal third of the ectodomain (helix f in domain iii, fig ) , which is involved in trimerization of the post-fusion structure [ , , ] . the longest α-helix predicted by phd in bvd g corresponds to this location ( fig. , aa - ). two shorter helices corresponding to helices g and h of vsv g are also predicted by the phd algorithm in bvd g (aa - , - ). with the exception of these α-helices, the ectodomains of bvd g are predicted to be comprised mostly of β-sheets, as is the case with other class iii vfp. another domain identifiable with computational tools in bvd g is the carboxyl terminal transmembrane anchor. tmpred, an algorithm that identifies possible transmembrane helices, assigns a significant score ( , > is statistically significant) to bvd g aa - , which suggests that this sequence includes the transmembrane anchor (violet cones). two other potential tm segments are present in bdv g ( - and - ). however, it is not likely that these regions are embedded in the membrane, either in g or gp because of the likely dicysteine bonding between cysteines and or other potential linkages involving cysteine . phd analyses also predict the presence of an α-helical stem domain with several aromatic aa prior to the transmembrane anchor (aa - ), a feature present in both class ii and iii vfp [ , ] . therefore, we have designated the sequence - as the transmembrane anchor, recognizing that the stem domain, which contains several aromatic amino acids, is likely to interface with the viral membrane. the fusion domains (referred to here as domain ii) of all class ii or iii vfp contain or fusion loops, which give significant scores on the wwihs [ ] . sequences with positive wwihs have a high potential to interface with or disrupt lipid membranes, and therefore are key features of vfp. another feature of the fusion domains of class ii and iii vfp is the presence of several dicysteine bonds, which stabilize the overall domain architecture. a region in bornavirus g (bvd aa - ) with cysteine residues, plus sequences with positive wwihs scores ( fig. , red, - , - ) is likely to represent the fusion domain of bdv g. sequence similarities between vsv and bdv g do not permit alignment by computational methods alone. however, using the regions of local structural similarity including the putative fusion domain/loops, extended αhelices and transmembrane domains, all of which are collinear, alignments between vsv and bdv g are proposed (figs. , ) . these alignments support assignment of a class iii domain architecture to bdv g. the proposed domains of bornavirus g are also collinear with analogous domains of herpesvirus gb, the other -albeit much longer -prototypic class iii vfp, as well as gp superfamily proteins encoded by baculoviruses and thogotoviruses (fig. ) . bdv g is more heavily glycosylated than vsv g, thov g or baculovirus gp , it is also more heavily glycosylated than gb of hsv- (fig. ) . cytomegalovirus (cmv) and epstein-barr virus (ebv) gb are closely related in sequence to hsv- gb. recent studies confirm that ebv gb is a class ii vfp [ ] , and it is likely that all herpesvirus gb are class ii vfp. cmv gb is glycosylated to a similar extent as bdv g, while ebv gb has an intermediate level of glycosylation. therefore, a high level of glycosylation does not preclude the possibility that bdv g is a class iii vfp. bdv g and other bornavirus g have consensus furin cleavage sites (rrrr) prior to the extended α helix (fig. , ) that are utilized in infected cells to cleave the proteins into two subunits gp and gp [ , ] . several alphaherpesviruses, such as varicella zoster virus (vzv), posses a furin cleavage site in the analogous location ( fig. dashed orange arrow). most beta and gamma-herpesviruses, collinear arrangement of similar structures in bdv g and vsv g figure collinear arrangement of similar structures in bdv g and vsv g. the post fusion secondary structure of vsv g as solved and numbered by roche and coworkers [ ] is depicted with α-helices as cylinders and β-sheets as arrows. the α-helices predicted by phd in bdv g are indicated similarly. β-sheets (t) and (u) of vsv g are not present in the protein data base structure ( cmz.pdb). in vsv g, α-helices predicted by phd are indicated by dashed boxes and predicted β-sheets are identified with dashed arrows. amino acids are numbered beginning after the putative signal sequences enclosed in parentheses. plum amino acids: n-glycosylation sites. sequences with significant wwihs scores in the fusion domain (ii) were identified by mpex and colored red. hydrophobic transmembrane domains (violet) were predicted using tmpred. a class iii domain nomenclature is used here that can apply to both class ii and iii vfp: domain i (green), domain ii (yellow), domain iii (blue), and stem domain (indigo). this unified nomenclature assigns domain ii (iv in the vsv g nomenclature of roche et al. [ ] , i in the hsv- gb nomenclature of heldwein et al. [ ] and ia and ib in the baculovirus nomenclature of kadlec et al. [ ] ) as the class iii fusion domain as in class ii vfps. in addition to minor adjustments in the ends of domains, the current class iii vfp numbering also combines two interacting domains into domain iii (i + ii in roche's vsv g nomenclature, iii + iv in heldwein's hsv- gb nomenclature and kadlec's baculovirus nomenclature). the domain numbering originally proposed is also indicated. ua represents "hinge" aa not assigned to domains in vsv g in the prior scheme. including cmv and ebv, have a furin cleavage site prior to the extended α-helix. therefore, possession of a furin cleavage site does not preclude the possibility that bdv gp is a class iii vfp. cysteine residues are usually the most conserved aa within a protein family because disulfide bonds between cysteines are critical determinants of secondary structure. the cysteines of class iii (and class ii) vfps determined by x-ray crystallography are arranged such that disulfide bonds are formed between cysteine residues within the same domain. to determine the plausibility of the proposed alignment, a model of bdv g scaffolded on the structure of vsv g in the post-fusion (low ph) configuration [ ] was constructed (fig. ) . the alignment between vsv and bdv g suggest that these vfps may have a similar structure. therefore, putative structures in bdv g are depicted as in vsv g. the proposed bdv g model is based principally on the structural predictions of phd, the most robust secondary structure prediction algorithm used. these results provide evidence that the cysteines in the putative fusion domain (domain ii) can potentially bond. such linkages can stabilize the fusion loops as occurs in both class ii and iii vfp. the dicysteine linkages are modeled such that all cysteine bonding occurs between the putative domains, as is the case with other class iii vfp. fig. . amino acids are numbered beginning after the putative signal sequences in vsv g, but at the beginning of the signal sequence of hsv- gb. arrows indicate g and gb truncations of the forms used for crystallography. solid lines represent cysteine bonding in vsv g, acmnpv gp , and hsv- and ebv gb [ , , , ] . black boxes represent hydrophobic regions, with violet representing the transmembrane anchor (tm). dashed lines represent potential cysteine bonding in bdv g, thov gp and cmv gb. dashed arrow is the location of a furin cleavage site in the alphaherpesvirus varicella zoster virus (human herpes virus ). solid arrows are furin cleavage sites in bdv g, cmv gb, and ebv gb. after cleavage by furin gp and gp of bdv appear not to be linked by covalent bonding, as the subunits disassociate during sds-page [ ] . therefore, we have not modeled any dicysteine linkages between the gp and gp subunits. there are plausible intradomain linkages that can form between each of the cysteines in bdv g. the bdv g structural model presented here is not intended as a definitive structural prediction. rather, there are many possible alternatives to the secondary and tertiary structures and the cysteine linkages of bdv g. for example, it is possible that in a minor subset of g may be cross-linked via cysteine binding such that the gp and subunits are covalently bound. the modeling does establish that feasible structures exist that are consistent with the secondary structure predictions and with the assignment of bdv g as a class iii vfp. proteomics computational analyses suggest that bornavirus g are class iii vfp. computational analyses and other methods predict that each of the major features common to class iii fusion proteins are present in bvd g, including internal fusion loops, an extended α helical domain, a stem domain and a carboxyl terminal transmembrane domain. these features of bdv g are located collinearly with those of vsv g, a prototypic class iii vfp [ , ] . on the basis of sequence similarities amongst g of members of the bornaviridae it is likely that all are class iii vfp. structural models including feasible cysteine linkage maps could be readily established for bdv g using the vsv g post-fusion structure as a scaffold. the fusion domains of bvd g, which we refer to here in a unified domain nomenclature as domain ii, appear to be stabilized by cysteine bonds and to contain one or more loops model of bdv g based on the x-ray crystallographic structure of vsv g figure model of bdv g based on the x-ray crystallographic structure of vsv g. the predicted structures of bdv g was fit to the post-fusion structure of vsv g [ ] . the post fusion secondary structure of vsv g as solved by roche and coworkers [ ] is depicted with α-helices as cylinders and β-sheets as arrows. β-sheets (t) and (u) of vsv g are not present in the protein data base structure ( cmz.pdb). sequences with significant wwihs scores were identified by mpex and filled red or outlined with red lines. hydrophobic transmembrane domains (violet) were predicted using tmpred as discussed in the text. secondary structures for bdv g were predicted by phd. orange/black lines: dicysteine linkages. black stick figures: n-glycosylation sites. with positive wwihs scores, features characteristic of the fusion domains of both class ii and iii vfp. differences between vsv and bdv g include the presence of a consensus furin cleavage site and a higher number of n-glycosylation sites in bdv g. however, many herpesvirus gb, which are class iii vfp, contain a furin cleavage domain prior to the extended α helix in domain iii and are more heavily glycosylated than vsv g. therefore, the presence of the furin cleavage site and high glycan content in bdv g does not preclude its putative inclusion in class iii. whether or not the secondary and tertiary folding of bdv gp conforms to the domain structure of class iii vfp will require x-ray crystallographic or other physical structural determinations. the three vfp classes for enveloped virus membrane glycoproteins were established based on structural similarities in the post-fusion configurations. therefore, it is likely that there is a common post-fusion (low ph) configuration of class iii vfp, and that bdv g has a post-fusion structure similar to vsv g. in contrast, the prefusion configurations of class i, ii and ii vfps are highly variable. the virion configuration of vsv g is homotrimer arranged in a tripod shape with the fusion domains corresponding to the legs of the tripod [ ] . no structural prediction of the prefusion configurations of bvd gp is possible. as in the case of class i fusion proteins, bdv g is cleaved by a cellular protease into two subunits [ , ] . it has been suggested that hydrophobic sequences following this cleavage site near the gp n terminus may function as a fusion peptide [ ] . the current modeling suggests that bornavirus g is not a class i vfp, and does not corroborate the existence of a "fusion peptide" in gp . the position of predicted α helices, dicysteine linkages and membrane interactive regions (sequences with positive wwihs scores) are not consistent with assignment of bdv g to class i. however, the concept that multiple domains of bornavirus g participate in fusion of viral and cellular membranes is consistent with current viral entry models. in class i vfp both the amino terminal fusion peptide, the pre-membrane aromatic domain and the tm cooperate to mediate lipid mixing. likewise, in class ii and iii vfp, fusion loops in domain ii, the stem domain, tm and other wwihs score positive sequences all appear to participate in fusion. several of the domains of gp and gp , particularly the putative fusion loops (domain ii), the sequences with positive wwihs scores in gp , and the stem and tm domain could cooperate or interact to mediate fusion and entry of bdv. bdv entry is via clathrin-mediated endocytosis, and the fusion between viral and cellular membranes occurs in the mildly acidic environment of the early endosome [ ] . the domains of bdv g involved in receptor recognition and cell entry have not been defined. gp sequences are capable of mediating attachment and entry in vsv pseudoparticles in the absence of gp [ ] . bdv-infected cells exhibit syncytium formation upon exposure to low-ph medium [ ] . this ph-dependent cell fusion event is likely mediated by gp since it is the only membraneanchored bdv glycoprotein found on the plasma membrane. these results suggest that both gp and gp are involved in membrane fusion, either cooperatively or in the case of cell surface expressed gp independently. an analogous situation may exist for hepatitis c virus (hcv). our previous modeling suggested that the envelope proteins of hcv split the duties performed by envelope proteins of other members of the flaviviridae, e of flaviviruses and e of pestiviruses [ ] . hcv e contains the fusion loops that are analogous to the domain ii fusion loops of e/e , while hcv e contains receptor binding domains analogous to domain iii of e/e , as well as the stem domain. the bdv cellular receptor(s) has not been identified. baculoviruses, which have a broad host range, may not possess a specific protein receptor. rather, it has been suggested that the fusion loops (domain ii) of baculovirus gp , a class iii vfp, may be the initial binding point with lipids of the target cell membrane [ ] . the host range of bornaviruses also appears to be broad. whether bornavirus g utilizes a specific protein receptor or bind to lipids or other ubiquitous components of cellular membranes remains to be determined. orthomyxoviridae, retroviridae, paramyxoviridae, filoviridae, arenaviridae, and coronaviridae and baculoviridae have members that encode class i vfp [ , , [ ] [ ] [ ] [ ] [ ] ] . flaviviridae, togaviridae, and bunyaviridae family members are known or appear to have members that encode class ii vfp [ , [ ] [ ] [ ] . if the current analyses are correct, bdv g joins rhabdovirus g, herpeviruses gb, thogotovirus g and baculovirus gp and as a class iii vfp. while convergence to common structures is possible, vfp of enveloped viruses may have evolved from a limited number of common progenitors. support for this hypothesis comes from the remarkable similarities in the post-fusion structures of the vfp in each class, even though the proteins differ dramatically in aa sequence. while, it is probable that other classes of vfp exist, there appears be a limited number of effective structures for virus-mediated membrane fusion. the bornaviridae are included with the rhabdovirdae and paramyxoviridae in the order mononegavirales. paramyxoviruses possess a class i vfp, whereas rhabdoviruses and, as suggested here, bornaviruses encode class iii vfp. there are several possibilities for how the glycoproteins of members of this order evolved. a g gene appears to have been present in the common ancestors of all members of the rhabdoviridae and bornaviridae. the similarities detected between rhabdovirus and bornavirus g are consistent with divergent evolution from a common progenitor, but sequence similarities are insufficient to establish a phylogenic relationship. therefore, it is possible that the class iii vfp of rhabdoviruses and bornaviruses were acquired by independent genetic events. an alternative suggested by kadlec et al. [ ] is that the three classes of vfp may have evolved from a common precursor (preclass i, ii, iii). this concept is based on morph videos and other analyses that reveal domain-specific folding and structural similarities amongst each of the three classes of vfp. if this is the case, mononegavirales evolution can be depicted as a "rooted" tree with the ancestral mononegavirus possessing a progenitor of class i, iii and likely ii vfp (fig. a) . alternatively, the vfp of paramyxoviruses could have been acquired independently from the acquisition of glycoproteins by rhabdoviruses and bornaviruses by horizontal gene transfer. this case can be depicted as an unrooted tree, in which glycoproteins were acquired after divergent of paramyxoviruses from rhabdovirus and bornaviruses (fig. b ). vfp are highly divergent at the primary sequence level. therefore, definitive statistical analyses of these possibilities are not possible at this time. the evolutionary origins of vfp, which display many common structural features, offer worthy challenges to computational biologists. in the absence of determinations by x-ray crystallography, structural models such as the one proposed here can provide useful hypotheses to guide experimental strategies for development of vaccines or drugs to prevent or treat infection by bornavirus infections. prior to the availability of x-ray structural data, several potent hiv- entry inhibitors were developed [ , ] based on the gallaher hiv- tm fusion protein model [ ] . fuzeon™ (dp ; t enfuvirtide), one of these peptides corresponding to a portion of the carboxyl terminal helix and the pre-anchor domain in this class i vfp, has been shown to substantially reduce hiv- load in aids patients, and is well-established in the treatment of hiv infection in the united states and european union [ ] . peptide entry inhibitors of viruses with class ii vfp have also been developed [ ] , and have also been described for the class iii vfp of herpesviruses (melnik et al., in preparation). given that bornaviruses cause a range of infectious neurological syndromes in warmblooded animals, with high mortality rates, veterinary applications of bornavirus entry inhibitors, should be investigated. confirmation of the still controversial proposal that bornaviruses cause neuropsyciatric disorders in acquisition of class i or iii vfp by members of the order mononegavirales figure acquisition of class i or iii vfp by members of the order mononegavirales. thick lines indicate primordial lineages and thin lines are lineages leading to contemporary viruses. the alternative in panel a shows a tree that is rooted, with the glycoproteins of all viral families evolved from a common class i, ii and iii progenitor. panel b shows another possible evolutionary tree, which is unrooted, and depicts independent acquisitions of class i vfp by an ancestral paramyxoviruses and class iii vfp of a common ancestor of rhabdoviruses and bornaviruses. a variation of this former tree (not shown) would involve separate acquisitions of class iii vfp by ancestral rhabdoviruses and bornaviruses. the biology of bornavirus recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent experimental induction of proventricular dilatation disease in cockatiels (nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus borna disease virus: implications for human neuropsychiatric illness human borna disease virus-infection and its therapy in affective disorders molecular biology of borna disease virus molecular biology of borna disease virus and persistence pathogenesis of borna disease in rats: evidence that intraaxonal spread is the major route for virus dissemination and the determinant for disease incubation cell-to-cell spread of borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein mechanism of borna disease virus entry into cells processing of the borna disease virus glycoprotein gp by the subtilisin-like endoprotease furin identification of the amino terminal subunit of the glycoprotein of borna disease virus maturation of borna disease virus glycoprotein inhibition of borna disease virus-mediated cell fusion by monoclonal antibodies directed against the viral glycoprotein n-terminal domain of borna disease virus g (p ) protein is sufficient for virus receptor recognition and cell entry structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution a general model for the transmembrane proteins of hiv and other retroviruses similar structural models of the transmembrane proteins of ebola and avian sarcoma viruses atomic structure of the ectodomain from hiv- gp the viral transmembrane superfamily: possible divergence of arenavirus and filovirus glycoproteins from a common rna virus ancestor model of the pre-insertion region of the spike (s ) fusion glycoprotein of the human sars coronavirus: implications for antiviral therapeutics detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein the envelope glycoprotein from tick-borne encephalitis virus at a resolution structure of the dengue virus envelope protein after membrane fusion conformational change and protein-protein interactions of the fusion protein of semliki forest virus proteomics computational analyses suggest that hepatitis c virus e and pestivirus e envelope glycoproteins are truncated class ii fusion proteins proteomics computational analyses suggest that the carboxyl terminal glycoproteins of bunyaviruses are class ii viral fusion proteins (beta-penetrenes) california serogroup gc (g ) glycoprotein is the principal determinant of ph-dependent cell fusion and entry crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g. science structure of the prefusion form of the vesicular stomatitis virus glycoprotein g crystal structure of glycoprotein b from herpes simplex virus proteomics computational analyses suggest that baculovirus gp superfamily proteins are class iii penetrenes the postfusion structure of baculovirus gp supports a unified view of viral fusion machines a time-efficient, linear-space local similarity algorithm prediction in d: secondary structure, membrane helices, and accessibility tmbase -a database of membrane spanning protein segments. bological chemistry hoppe-seyler membrane protein explorer version experimentally determined hydrophobicity scale for proteins at membrane interfaces the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein e mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptadrepeat region of hepatitis c virus glycoprotein e structure of a trimeric variant of the epstein-barr virus glycoprotein b cell entry of borna disease virus follows a clathrin mediated endocytosis pathway that requires rab and microtubules identification of an n-terminal trimeric coiled-coil core within arenavirus glycoprotein permits assignment to class i viral fusion proteins characterization of a putative cellular receptor for hiv- transmembrane glycoprotein using synthetic peptides a synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry peptide inhibitors of dengue virus and west nile virus infectivity the authors declare that they have no competing interests. ceg performed sequence alignments, and assisted in preparation of figures. rfg wrote the manuscript. both authors read and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge key: cord- - aj d authors: fongaro, gislaine; nascimento, mariana a do; rigotto, caroline; ritterbusch, giseli; da silva, alessandra d’ a; esteves, paulo a; barardi, célia r m title: evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: aj d background: human adenoviruses (hadvs) are the second-leading cause of childhood gastroenteritis worldwide. this virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially uv light inactivation. molecular techniques, such as pcr-based methods (polymerase chain reaction), are commonly used to detect and identify viral contamination in water, although pcr alone does not allow the discrimination between infectious and non-infectious viral particles. a combination of cell culture and pcr has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (cpe) in cell culture. this study aimed to assess the integrity and viability of human adenovirus (hadv) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in florianópolis, santa catarina island, brazil: peri lagoon water, spring source water, and water from the public water supply system. methods: water samples were collected, concentrated and hadv quantified by real-time pcr. viral integrity was evaluated by enzymatic assay (dnase i) and infectivity by plaque assay (pa) and integrated cell culture using transcribed mrna (icc-rt-qpcr). samples containing particles of infectious hadv were selected for sequencing and molecular characterization. results: the analyzed sites contained , and % undamaged hadv particles (defined as those in which the genetic material is protected by the viral capsid) at peri lagoon, spring source water and public supply system water, respectively. of these, % of the particles (by pa) and % (by icc-rt-qpcr) hadv were shown to be infectious, due to being undamaged in peri lagoon, % (by pa) and % (by icc-rt-qpcr) in spring source water and % (by pa) and % (by icc-rt-qpcr) in the public water supply system. icc-rt-qpcr, a very sensitive and rapid technique, was able to detect as low as × ( ) hadv genome copies per milliliter of infectious viral particles in the environmental water samples. the molecular characterization studies indicated that hadv- was the prevalent serotype. conclusions: these results indicate a lack of proper public health measures. we suggest that hadv can be efficiently used as a marker of environmental and drinking water contamination and icc-rt-qpcr demonstrated greater sensitivity and speed of detection of infectious viral particles compared to pa. waterborne viral infection is one of the most important causes of human morbidity, and related diseases continue to have public health and socioeconomic implications worldwide. according to existing reports in the literature, there are hundreds of different types of human viruses present in human sewage, which, if improperly treated, can become a source of contamination in drinking and recreational water [ ] . human adenoviruses (hadvs) are the second-leading cause of childhood gastroenteritis worldwide. this virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially uv light inactivation [ ] [ ] [ ] . along with other pathogens, hadv is on the usepa (united states environmental protection agency) candidate contaminants list [ ] as they are important human pathogens that can be transmitted by water consumption and water spray (aerosols). molecular techniques, such as pcr-based methods (polymerase chain reaction), are commonly used to detect and identify viral contamination in water, particularly those viruses that do not multiply easily in cell culture [ ] . the concentration of pcr inhibitors in environmental water samples and the ability of such techniques to detect naked nucleic acids from non-infectious pathogens may represent a limitation for the use of pcr as a detection tool. in addition, pcr alone does not allow the discrimination between infectious and non-infectious viral particles [ , ] . a combination of cell culture and pcr has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (cpe) in cell culture [ ] . integrated cell culture pcr (icc-pcr) has the benefits of cell culture and pcr and attempts to compensate for several cell culture disadvantages, such as its time-consuming nature and limited detection sensitivity [ ] . however, this method has a disadvantage in that detection of nucleic acids of inactivated viruses from environmental samples adsorbed onto cell receptors without cell infection can result in false positives [ ] . therefore, it is necessary to confirm infectious viruses by assaying infection of the permissive cells and subsequent transcription of viral mrna. thus, the detection of viral mrna in cell culture indicates the presence of infectious viral particles [ ] . plaque assay is another method that can be used to infer the ability of viruses to infect and cause lysis in a cell monolayer [ ] . enzymatic assays can also be used to check the integrity of the viral capsid by using dnase or rnase nucleases. genetic material that is not protected by a viral capsid will be degraded by these nucleases [ ] . in this study, we aimed to assess the integrity and viability of human adenovirus (hadv) detected in environmental water samples and also to use molecular characterization to evaluate circulating strains. the efficiency of the viral recovery using the concentration method described by katayama et al. ( ) [ ] was measured. when analyzing the samples from all sites ( , , and ), the recovery rate was approximately %, as evaluated with both pfu and gc units. the pa value (pfu/ml) equivalence, when compared to icc-rt-qpcr values (gc/ml) immediately after the concentration of water samples and after a series of decimal dilutions ( to - ), is shown in figure . the average equivalence of a pfu unit for gc was . logs, which means that pfu is equivalent to gc. this proportionality was confirmed via statistical testing (linear regression test) conducted with graphpad prism version . (usa). the icc-rt-qpcr assay was more sensitive than the pa in the detection of hadv, demonstrating a detection limit of × gc/ml (decimal dilution - ), while the pa had a detection limit of × pfu/ml (decimal dilution - ) (figure ). twenty-five of ( . %) samples contained undamaged hadv particles and / ( . %) contained infectious hadv particles. the rate (%) and the mean quantification of the undamaged and infectious hadv particles, per site, are listed in table . all positive sites ( , , and ) samples contained undamaged and infectious viral particles. not all samples with undamaged virus particles were shown to be infectious, but this difference was not statistically significant (p > . , anova in graphpad). the icc-rt-qpcr was more sensitive than the pa for detecting infectious particles in environmental water samples (table and figure (a, b, c)). the results indicate that of lagoon water (site ) samples contained undamaged viral particles. these all contained infectious particles detectable by icc-rt-qpcr and samples contained infectious particles detectable by pa. in addition, of source water (site ) samples contained undamaged viral particles. of these, only samples contained infectious particles detectable by icc-rt-qpcr and sample contained particles detectable by pa. lastly, of public supply system water (site ) samples contained undamaged viral particles. of these, contained infectious particles detectable by icc-rt-qpcr and contained infectious particles detectable by pa. these results are presented in figure (a, b, and c). a total of samples ( from lagoon water, from spring source water, and from public supply system water) that contained infectious or undamaged hadv particles were sequenced for molecular characterization. the obtained sequences were compared with the ones already deposited in ncbi and presented - % identical to sequences of hadv serotype of subgroup c. the resulting assembled phylogenetic tree is shown in figure . when compared, the amino acid sequences of samples contained an amino acid substitution. this has been observed in the lagoon water samples (samples , , , , and ), the spring water samples (samples , and ) and the public water supply system samples (samples , and ). the effect of these substitutions was evaluated using the phyre program, which indicated that the substitutions did not alter the secondary and tertiary structures of the hadv hexon protein (data not shown). adenoviruses are among the most studied groups of potential viral indicators of water quality [ ] , this is due to the huge number of viral particles that are consistently found in environmental waters worldwide [ ] . with that in mind, this study presents data on the integrity, infectivity and molecular characterization of hadv in water samples (lagoon, spring source and public supply system) collected over a period of year from florianópolis, which is located in southern brazil. in many countries, the water quality is evaluated according to bacteriological standards, even though bacterial contamination is not correlated with the presence of human enteric viruses [ ] . the impact caused by water contamination of enteric viruses has been less studied than the impact caused by bacteria and protozoa. due to the high cost of equipment and laboratory reagents, it can be difficult to detect viral agents in environmental samples, as they sometimes present in very low concentrations in the sampled material or because some viruses are not detectable in cell culture assays [ ] [ ] [ ] [ ] . in this study, the filtration, concentration and elution method using a negatively charged membrane was applied to concentrate viruses from water samples. this method has been described as a useful tool to recover enteric viruses from environmental samples and allows the detection of such viruses by molecular methods [ ] [ ] [ ] [ ] [ ] [ ] . as already reported in the literature, it is important to evaluate viral integrity (state of preservation of viral particles, with undamaged particles being defined as those in which the genetic material is protected by viral capsid) [ ] and infectivity, (ability of the virus to replicate in permissive cells) [ ] , instead of only evaluating the presence or absence of the viral genome. therefore, the processed samples were used to evaluate viral integrity (via enzymatic assay with dnase i) and infectivity (via pa and icc-rt-qpcr-et) of hadv present in the collected water samples. the majority of samples evaluated in this study contained intact hadv particles, but this result does not mean that those particles were infectious, as factors such as temperature, ph and uv radiation are known to cause conformational changes in the viral capsid, resulting in figure undamaged and infectious hadv particle: average number of undamaged and infectious hadv particle detected in water samples: a) lagoon water b) spring source water, and c) public supply system water. in black bar: undamaged particles; in gray bar: infectious particle, measured by icc-rt-qpcr assay, and in line: infectious particles, measured by (pa). loss of infectivity [ ] . on the other hand, hadv has the ability to form aggregate particles in the water that protect the individual viral particles from inactivating factors [ ] . all the samples containing particles of infectious hadv detected by pa were also detected with icc-rt-qpcr, but the reverse was not true. icc-rt-qpcr demonstrated greater sensitivity and speed of detection of infectious viral particles compared to pa. icc-rt-qpcr had a detection limit of × gc/ml at h. specificity and sensitivity are also important aspects to consider, as the icc-rt-qpcr relies on mrna and thus avoids false negatives or positives [ , ] . the integrity of hadv particles was positively correlated with their infectiousness, as evaluated by icc-rt-qpcr, which corroborates other studies that have reported that enzymatic techniques are a valuable alternative when making inferences about potential viral infectivity, as they avoid laborious cell culture techniques and not all viruses can replicate in cell culture [ ] . hadv serotypes that are responsible for gastroenteritis are hadv and that are usually difficult to propagate in cell culture, so the use of enzyme tests can infer on viral infectivity, being a less laborious than techniques in cell culture [ ] . molecular and bioinformatics studies have both demonstrated higher efficiency in characterization, as in the discovery of new virus subtypes [ , ] . hadv- is commonly found throughout the world. the virus subtype causes diseases that affect the upper respiratory tract, particularly in children [ , ] . the results of the hadv characterization performed in the present study, by sequencing and molecular characterization, indicated the prevalence of hadv, serotype subgroup c in the samples evaluated. the viruses that belong to hadv subgroup c (c -c -c -c , and c ) are known to cause respiratory viral infections [ ] . our findings corroborate those of other studies that reported the prevalence of hadv- in the aquatic environment and in stool samples [ ] [ ] [ ] . taken together, these studies and others indicate that hadv- is one of the serotypes most commonly excreted by humans [ ] , suggesting that this virus is intermittently excreted in the feces of most individuals, even if they are asymptomatic [ ] . some substitutions in the hadv amino acids sequences were observed and analyzed in this work. other recent studies have observed substitutions of amino acids in respiratory hadv, suggesting that the substitutions are related to respiratory-tissue viral tropism [ , ] . fecal pollution of drinking water is a primary health concern. the incidence of infectious hadv in water samples indicates a contamination of these sources with human effluents. these results indicate a lack of proper public health measures, justifying the urgent necessity of adding methods for the detection, removal and inactivation of such viruses during the water treatment process. furthermore, we suggest that hadv can be efficiently used as a marker of environmental and drinking water contamination. two liters of surface water samples were collected monthly for one year from three locations on florianópolis, the capital of the state and island of santa catarina in southern brazil. a total of samples were collected. site (peri lagoon), samples were collected from june to may . this water source is used by the local water company plant and provides potable water for more than , inhabitants. site (spring source water -untreated) was collected from february to january . site (public supply system waterspring source water chlorinated for human consumption) was collected from february to january . the collective supply systems are controlled and regulated by the ordinance of the federal ministry of health of brazil (ms / ) [ ] . sample concentration was performed as described by katayama et al. ( ) [ ] . briefly, this method consists of filtration, concentration, and elution of viral particles in water samples through a negatively-charged membrane. the final sample was approximately ml before using a centriprep reconcentrated ym ® (millipore), resulting in a final volume of approximately ml, after centrifugation at , × g for min at °c. the concentrated samples were stored at − °c until further analysis. the extraction of viral nucleic acid was performed using a commercial rtp dna/rna virus® ii mini kit (invitek), according to the manufacturer's instructions. nucleic acid was eluted in a μl elution buffer and stored at − °c until further analysis. a reverse transcriptase (rt) reaction was performed to generate cdna from mrna, using an rt enzyme and random primers (sensiscript rt kit -qiagen®). quantitative pcr was performed as described by hernroth et al. ( ) [ ] . all amplifications were done in a stepone plus® real-time pcr system (applied biosystems). each sample was analyzed in triplicate. for each plate, four serial dilutions of standard were run in triplicate for each assay and the genome copies (gc) were measured. ultra-pure water was used as the non-template control for each assay. to infer the presence of undamaged viral particles, hadv-positive samples, as detected by qpcr, were treated with dnase i, as described by viancelli et al. ( ) [ ] . to verify potential inhibitors of dnase i present in the sample matrix, a known amount of previously inactivated hadv- ( h at °c and min under uv irradiation) was added in concentrated samples (previously hadv- negative) of all sites and in nuclease-free water (nfw), as a control. the reactions were performed using u of dnase (sufficient quantity to degrade % of dna added), × buffer and μl of sample or nfw and incubated for min at room temperature, with the intention of degrading all free genetic material. then, the enzyme was inactivated with edta mm and incubated for minutes at °c. these treated samples/nfw were then subjected to nucleic acid extraction and qpcr, as described previously. for virus viability assays of environmental water samples, hadv was propagated in a continuous line of a cells (permissive cells derived from human lung carcinoma cells, european collection of cell cultures). these cells were kindly donated by dr. rosina gironès from the university of barcelona, spain. to infer the presence of infectious hadv particles, water samples were previously treated with u/ml penicillin, μg/ml streptomycin and . μg/ml amphotericin b. the treated samples were inoculated ( . ml) at a non-cytotoxic dilution, in triplicate, in a cells for the plaque assay, as described by cromeans et al. ( ) [ ] and rigotto et al. ( ) [ ] . the cells were incubated for h at °c with rotation every min. the cells were then removed and overlaid with . % bacto-agar (previously melted) in high glucose dmem x (high-glucose dulbecco's modified eagle's medium) containing % fbs, . mm sodium pyruvate, u/ml penicillin, μg/ml streptomycin and mm mgcl . the samples were then incubated at °c for days. then, the agar overlay was removed, and the cells were stained with % gram's crystal violet and the plaques counted macroscopically. to quantify the number of infectious hadv particles present in the water samples, an icc-rt-qpcr assay (integrated cell culturepreceded by reverse transcriptase and qpcr) was conducted. the protocol was adapted from rigotto et al. ( ) [ ] and ko et al. ( ) [ ] and aims to access viral mrna following viral replication in cells. water samples, in a non-cytotoxic dilution, were inoculated in triplicate in a cells for the icc-rt-qpcr assay. after h of incubation at °c with rotation every min, the inoculum was removed and the cell layers were overlaid with high-glucose dulbecco's modified eagle's medium (dmem) before being incubated at °c for h. after incubation as described previously, the supernatant was recovered and . ml was used for genetic material extraction, as described above. immediately after the extraction of the total nucleic acids, enzymatic treatment, with dnase i, was conducted in order to degrade the dna in the sample tested. then a reverse transcriptase reaction (rt) was used to generate cdna from viral mrna. the quantification of infectious particles of hadv was performed with qpcr, as described above. for each site analyzed, to determine the efficiency of the viral recovery by the concentration method, a known concentration of hadv was spiked into l of each water matrix (previously negative for hadv by qpcr). the samples were concentrated, and the viral recovery was assessed by plaque assay (pa) and by integrated cell culture-rt-qpcr (icc-rt-qpcr), as described below. to determine the equivalence of the pa viral quantification to the icc-rt-qpcr value, and also to confirm the ability of these techniques to detect infectious hadv in each water matrix, decimal dilutions ( to - ) of concentrated seeded water samples were created and analyzed. samples containing particles of infectious hadv were selected for sequencing and molecular characterization. the primers described by allard et al. ( ) [ ] , directed to a region of the viral genome coding for the capsid hexon protein ( - bp position in hadv genome) common to all hadvs, were used for pcr, generating a product of approximately bp. the amplicons were purified with exosap-it (ge healthcare uk ltd, buckinghamshire, uk). ultra-pure water was used as the non-template control for each assay. sequencing was carried out in a genetic analyzer with the big dye terminator v . cycle sequencing kit (applied biosystems) and following the manufacturer's protocol. each product was sequenced four times in both directions. the quality of dna sequences was checked and overlapping fragments were assembled using the bioedit package . . [ ] , and contigexpress (informax, inc.). after the alignment, the sequences were compared with the ones deposited in genbank using the blast tool [ ] and the molecular characterization was conducted with mega . software [ ] . the homology analyses (evolutionary history) were inferred by using the maximum likelihood method based on the kimura parameter model and calculated using pairwise deletion. bootstrap was resampled as a test of phylogeny using replications [ ] . the statistical analyses were performed using graphpad prism version . (usa). a pearson correlation and linear regression test, anova test and student's t test were performed (p < . ). enteric viruses of human and animals in aquatic environments: health risks, detection, and potential water quality assessment tools waterborne adenovirus: a risk assessment inactivation of feline calicivirus and adenovirus type by uv radiation comparative inativation of enteroviruses and adenovirus by uv light usepa -united states environmental protection agency: contaminant candidate list -ccl bofill-mas s: molecular detection of pathogens in water -the pros and cons of molecular techniques real-time pcr quantification of human adenoviruses in urban rivers indicates genome prevalence but low infectivity evaluation of pepper mild mottle virus, human picobirnavirus and torque teno virus as indicators of fecal contamination in river water pretreatment to avoid positive rt-pcr results with inactivated viruses rt-pcr amplification detects inactivated viruses in water and 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samples collected from swine manure treatment systems development of plaque assays for adenoviruses and survival of adenovirus types and in surface and ground waters measured by a plaque assay detection of adenoviruses in shellfish by means of conventional-pcr, nested-pcr, and integrated cell culture pcr (icc ⁄ pcr) detection of adenoviruses in stools from healthy persons and patients with diarrhea by two-step polymerase chain reaction bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt gapped blast and psi-blast: a new generation of protein database search programs mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods bootstrap confidence levels for phylogenetic trees evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays written informed consent was obtained from the patient for publication of this report and any accompanying images. the authors declare that they have no competing interests.authors' contributions gf, man and cr design and carried out the research, gr, ads and pae contributed in the sequence analysis, and gf and crmb wrote the paper. all authors read and approved the final manuscript. key: cord- -rfwzj at authors: riaz shah, shahida amjad; idrees, muhammad; hussain, abrar title: hepatitis g virus associated aplastic anemia: a recent case from pakistan date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: rfwzj at background: aplastic anemia (aa) is a serious and rare disorder characterized by a hypocellular bone marrow. hepatitis associated aplastic anemia (haaa) is a variant of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. several reports have noted an association between hgv and hepatitis-associated aplastic anemia besides other hepatitis causing viruses. case presentation: a female girl of age year with a history of loose motion for one month, vomiting for last days and poor oral intake for last few days is reported here. the physical examination presents fever, pallor whereas bleeding, hepatomegaly, splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, splenomegaly and lymphodenopathy. the laboratory investigation parameters were: haemoglobin . g/l, total leucocytes count . , neutrophils . %, absolute reticulocyte count . %, monocytes . %, red cell count . mil/ul, picked cell volume (pcv) . %, mean corpuscular volume (mcv) fl, mean corpuscular hemoglobin (mch) . pg. the liver enzymes were alanine aminotransferease (alt) iu/l, aspartate aminotransferase (ast) iu/l. serologic and molecular tests for hepatitis a, b, c, d, e, ttv, b were negative, whereas hgv rna pcr test was found positive for hepatitis g virus. the bone marrow aspirate and trephine biopsy examination revealed hypo- cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis. conclusion: haaa is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. our finding suggests the involvement of hgv in the development of aplastic anemia. in patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by rt-pcr and if possible by bone marrow biopsy. hepatitis g virus was reported first time has a non-a-e hepatitis and placed as flavivirus [ ] .the induction of this new agent in the family of hepatitis has attracted significant attention because of its etiology [ ] . hepatitis g virus has been marked as a cause of non-a through e acute viral hepatitis and sharp liver failure. aplastic anemia complicating hepatitis is an uncommon but well recognized phenomenon. hepatitis associated aplastic anemia is a severe disorder with a high mortality ( %) [ ] .hepatitis associated aplastic anemia (haaa) is a deviation of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. the marrow failure can be severe and is usually lethal if untreated. lorenz and quazier has documented first time haaa in two case back in [ ] , by more than cases had been reported [ ] . adil et al has reported, severe aplastic anaemia (saa) . %, very severe (vsaa) in . % of patients of aplastic anemia cases [ ] . a number of reports have mentioned alliance between hgv and haaa [ ] [ ] [ ] [ ] . number of haaa cases with a history of multiple blood transfusions has been reported [ , ] . crespo et al has documented a case of year old man have community acquired hgv that later progress into severe aplastic anemia, point out hgv for both hepatitis and aplastic anaemia. however greater number of serum samples are needed to prove the association of hepatitis g virus and aplastic anaemia [ ] . moatter et al. has reported / patients of haemodialysis with raised liver enzyme, reduced platelet count and / patients of polytransfused b-thalassemia major children infected with hgv rna form pakistan [ , ] . in this study well characterized samples of aplastic anaemia patients before blood tranfusion were included. these characteristics include history, physical examination, haematological investigation, bone marrow aspirate and trephine biopsy examination, liver function test (lfts), renal parameters, viral profile and abdominal ultrasonography. the diagnosis of ha-aplastic anemia was made on the basis of hepatitis (elevated serum aminotransferase enzymes, jaundice, absolute neutrophils counts, platelet counts and reticulocytes. all the samples were checked for serological marker of hav, hbv, hcv, hdv, hev, hgv,ttv and b . one of samples from patients with ha-aplastic anemia has hepatitis g associated aplastic anaemia with positive hgv rna. a female girl of age year is reported here. the patient had a history of loose motion for one month, vomiting for last days and poor oral intake for last few days. the physical examination presents fever, pallor whereas bleeding, hepatomegaly, splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, splenomegaly and lymphodenopathy. the laboratory investigation parameters were: haemoglobin . g/l, total leucocytes count . , neutrophils . %, absolute reticulocyte count . %, monocytes . %, red cell count . mil/ul, picked cell volume (pcv) . %, mean corpuscular volume (mcv) fl, mean corpuscular hemoglobin (mch) . pg. the liver enzymes were alanine aminotransferease (alt) iu/l, aspartate aminotransferase (ast) iu/l. serologic and molecular tests for hepatitis a, b, c, d, e, ttv, b were negative, whereas hgv rna pcr test was found positive for hepatitis g virus. the bone marrow aspirate and trephine biopsy examination revealed hypo-cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis. flaviviruses belong to enveloped viruses with a single positive sence rna about kb. hepatitis g virus medium of transmission mostly through blood. the possible roel of hepatitis g virus infection in the pathogenesis of rare non-liver disease has been suggested but need to be recognized. by some unknow reseason aplastic anemia some time proceded by hepatitis. few reports have analysed the role of hgv in the development of haaa [ , , ] . crespo et al in print a case through negative serological markes for hav, hbv, hcv, hev, hypoplastic marrow low platelet and white cell counts but detected hgv-rna before any blood transfusion [ ] . byrnes et al has described hepatit g virus positive case of year old man before the use of medication, blood tranfusion or intravenous drug abuse [ ] . zaidi et al, reported a year male before blood transfusion with positive hgv by rt-pcr and suggested that in the absence of any other clincial manifestions the possible infectious agent may be hgv for hepatitis g virus associated aplast anaemia [ ] . in the list of studies of hepatitis g virus associated aplastic anaemia before blood transfusion we report a case of years female girls. whereas some reports confirms the presence of hgv viraemia in the patient aplastic anaemia after blood transufion [ , , , , [ ] [ ] [ ] . but however concluded that hgv viraemia is frequent in patients with aplastic anaemia [ ] . kiem et al reported similar finding after logistic regression analysis, that hgv rna in transfused patients was . times higher compare to untransfused patients (p = . ). this implicates transfusion as major source of hgv with aplastic anaemia [ ] . the published literature point out that studies must be performed on many more aplastic anaemia patients prior to blood transfusion [ ] . however, to find such patients in large number are not normally avaible to study, so far individual cases are reported. the ideal case regarding hepatitis g associated aplastic anaemia are pre blood transfusion. in conclusion, haaa is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. our finding suggests the involvement of hgv in the development of aplastic anemia. in patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by rt-pcr and if possible by bone marrow biopsy. written informed consent was obtained from the patients a copy of which is available for review by editor-in-chief of this journal. authors' contributions sars aided in acquistion of data that was included in this case report and drafted the manuscript. mi aided in acwuision and interpretation of the data analysed. ah helped in statistical analysis of the data and in editing of this manuscript. all authors have read and approved the final version of this manuscript. identification of two flavivirus-like genomes in the gb hepatitis agent hepatitis g virus panmyelopathy following epidemic hepatitis aplastic anemia following viral hepatitis: report of two fatal cases and literature review epidemiological features of aplastic anaemia in pakistan aplastic anemia and viral hepatitis aplastic anaemia after hgv infection hepatitis g-associated aplastic anaemia molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent hepatitis-associated aplastic anemia hepatitis-associated aplastic anemia hepatitis g virus infection as a possible causative agent of community-acquired hepatitis and associated aplastic anaemia hepatitis g virus in karachi, pakistan. lancet prevalence of hepatitis g virus in pakistani children with transfusion dependent beta-thalassemia major prevalence of hepatitis g virus in patients with aplastic anemia the incidence of transfusion-associated hepatitis g virus infection and its relation to liver disease hepatitis gb virus c genome in the serum of aplastic anaemia patients receiving frequent blood transfusions prevalence of gbv-c/hgv, a novel 'hepatitis' virus, in patients with aplastic anaemia hepatitis g virus: molecular organization, methods of detection, prevalence, and disease association hepatitis g virus associated aplastic anemia: a recent case from pakistan the authors declare that they have no competing interests. key: cord- -rya w sh authors: kang, xiaoping; li, yuchang; fan, li; lin, fang; wei, jingjing; zhu, xiaolei; hu, yi; li, jing; chang, guohui; zhu, qingyu; liu, hong; yang, yinhui title: development of an elisa-array for simultaneous detection of five encephalitis viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: rya w sh japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), and eastern equine encephalitis virus (eeev) can cause symptoms of encephalitis. establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. currently, there are still no multiple antigen detection methods available clinically. an elisa-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. an elisa-array method for the simultaneous detection of five encephalitis viruses was developed in this study. seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the elisa-array. the elisa-array assay is based on a "sandwich" elisa format and consists of viral antibodies printed directly on -well microtiter plates, allowing for direct detection of viruses. the developed elisa-array proved to have similar specificity and higher sensitivity compared with the conventional elisas. this method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. the results demonstrated that the developed elisa-array is sensitive and easy to use, which would have potential for clinical use. japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), eastern equine encephalitis virus (eeev), sindbis virus(sv), and dengue virus(dv) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [ ] . establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. currently, elisa and ifa are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [ , ] . there are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [ ] [ ] [ ] . however, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [ ] [ ] [ ] [ ] . liew [ ] validated one multiplex elisa for the detection of antigens; anderson [ ] used microarray elisa for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that elisa-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. however, the application of elisa-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. in this study, we developed an elisa-based array for the simultaneous detection of five encephalitis viruses. seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an elisa-array assay. the assay was validated using cultured viruses and inoculated chicken eggs with patient sera. the results demonstrated that this method combined the advantage of elisa and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. monoclonal antibodies were prepared from hybridoma cell lines constructed by prof. zhu et al. purification was conducted by immunoaffinity chromatography on protein g affinity sepharose [ ] . specific monoclonal antibodies ( d against jev, b against tbev, f against sv, b against serotype dv, f against serotype dv, e against eeev, and a against flavivirus) were selected for this study. all of the antibodies were raised according to standard procedures. using d , b , f , b , f , and e as capture antibodies, detection antibodies ( a , f , and e ) were coupled to biotin-nhs ester(pierce, germany) at °c for h according to the manufacturer's instructions. unincorporated biotin was removed by desalt spin column (pierce). immunologic reactions were reported by streptavidin-hrp (cwbio, beijing, china) and super signal elisa femto maximum sensitive substrate. purified goat-anti mouse antibody was used as a positive control. jev and dv were cultured in c / cells; sv, tbev, and eeev were cultured in bhk- cells. the culture of tbev and eeev was conducted in biosafety level facility, however, jev, dv and sv were conducted in biosafety level facility. viral titers were determined by the % tissue culture infectious dose (tcid ) method. all the cultures were inactivated by . % β-propionolactone at °c overnight, then °c for h to decompose β-propionolactone. antibodies were spotted using a biodot machine (bd ;california, usa) on elisa plates ( nl/dot). the plates were blocked with % bsa-pbs in °c for h, followed by washing times with pbs containing . % tween- for min each. then, the plates were dried, sealed, and stored at °c before use [ ] . when spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the elisa-array assay. the optimization was evaluated by dot morphology and signal intensity. the tested spotting buffers included × phosphate buffer saline (pbs), pbs + % glycerol, and × pbs + % glycerol+ . % triton-x . a range of monoclonal antibody concentrations ( . , . , . , . , and . mg/ml) were compared. following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, hpr-labeled avidin, and substrate, followed by signal evaluation. antigen binding was performed in pbs(containing . % tween- and % fcs) at °c for h, followed by washing times( × pbs containing . % tween- ). incubation of elisa plates with biotinylated detecting antibody cocktails was performed in pbs (containing . % tween- and % fcs) at °c for h. after washing, specific binding of the detecting antibodies was reported by streptavidin-hrp and stained with super signal elisa femto maximum sensitive substrate (thermo scientific, rockford, usa) [ , , ] . visualization of the plate was performed in ae cool ccd image analyzer(beijing bgi gbi biotech company., ltd, china). the signal intensity and background of each spot was read out and recorded with "monster"software. the positive signals were defined as a signal value > and a signal value (sample)/signal value (negative) > . the identical antibodies used in the elisa-array format were also tested in a conventional elisa format to determine the difference in sensitivity and specificity of the two methods. the conventional elisas were performed at the same time as the elisa-array assays to ensure similar reaction conditions. the conventional elisas were performed in an identical maner to the elisa-array, except that antibodies were coated at a concentration of μg/ml in pbs (ph . ), and substrate tmb was used instead of super signal elisa femto maximum sensitive substrate [ , ] . three serum samples were collected from patients with nervous system symptoms and histories of tick bites. the serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. days later, the liquid was collected and divided into two portions (one for inactivation and one for rna extraction). the rna and inactivated samples were stored at - °c before use. rna was extracted from the inoculated chicken eggs using a rneasy mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's instructions. all rna extraction procedures were conducted at bsl- facilities. the primers and probes were used as previously described [ ] . the real-time rt-pcr was conducted with a quti-teck q-rt-pcr kit (qiagen inc,). the reaction consisted of μl of × reaction buffer ( . μl reverse transcription enzyme, and nmol/l primers and probes). rna and deionized water were added to a final volume of μl. pcr was performed with a lightcycler . (roche, switzerland) [ ] . optimization of the elisa-array assay the spotted array layout is depicted in figure and the efficacy of three different spotting buffers on the quality of the printed elisa-arrays were investigated by spot morphology observation and signal intensity comparison. the spotting concentration of the capture antibodies varied from . to . mg/ml (each was serially diluted -fold). the efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. figure illustrates the array layout and figure demonstrates the result of the three spotting buffers and spot concentration of antibody b by tbe virus culture detection. cross reaction detection was also conducted by applying jev, yf, and dv cultures. spot morphology observation (figures a, b , and c) demonstrated that spotting buffer containing pbs with % glycerol produced tailed spot morphology; buffers containing pbs alone and pbs with % glycerol + . % triton-x gave good spot morphology (round and full). buffers containing pbs with % glycerol and pbs with % glycerol+ . % triton-x produced higher signal intensities than pbs alone. thus, pbs with % glycerol+ . % triton-x was adopted as the optimized spotting buffer for subsequent experiments. simultaneously, the spot concentration evaluation suggested that . mg/ml was optimal. at this concentration, the signal intensity was higher and the cross-reaction did not appear (figure d ). consequently, spotting concentration optimization of other capture antibodies ( d , f , e , and b ) demonstrated that . mg/ml was also suitable(data not shown). the optimized elisa array layout is shown in figure , which was applied in the following experiments. successful detection of viral pathogens requires a test with high sensitivity and specificity. to evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. by testing serially-diluted viral cultures, including dv- , dv- , jev, tbe, sv, and eeev, the sensitivity of elisaarray and the identical conventional elisa were compared ( table ). the detection limit of the two methods was compared and demonstrated. the cross-reactivity test was conducted using bhk- and vero cell lysate, yellow fever virus (yfv) cultures ( × tcid /ml, west nile virus(wnv) cultures( × tcid /ml), and western equine encephalitis virus( × tcid /ml). the results demonstrated that neither the elisa-array nor traditional elisa displayed cross-reactivity. equal volumes of cultured tbev, jev, dv- , dv- , sv, and eeev were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. a cocktail of biotin conjugated antibody ( a , e , and f ) was used in all tests. the results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (figures and ) . chicken eggs inoculated with infected human serum were used for validation of the elisa-array assay. all samples showed high reaction signals with capture antibody b , which was specific for tbev ( figure b ). the elisa-array assay suggested that the three patients were all infected with tbev. to verify the results tested by elisa-array, rna extracted from chicken eggs was applied to a real time-rt-pcr assay using primers and probes targeting tbev. the results were also positive (figure a) . the consensus detection results confirmed that the elisaarray assay was reliable. to be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [ , , ] . multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. thus, an elisa-array, which combines the advantages of elisa and protein array, meets the above requirements. it has been reported that an elisa-array has been used in the diagnosis of cancer and auto-allergic disease [ , ] ; however, no study has reported the detection of viral pathogens. in this study, we developed a multiplex elisa-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. the production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [ ] . cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. first, we prepared and screened monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. then, the antibodies were screened by an elisa-array with a double-antibody sandwich elisa format. the antibodies which produced cross-reactivity and low-positive signals were excluded. finally, six antibodies were selected as capture antibodies. another monoclonal antibody, a , which could specifically react with all viruses in the genus flavivirus was used for detecting antibody against dv, jev, and tbev. for the detection of eeev and sv, although the detecting and trapping antibodies were the same ( f and e , respectively), the antibodies produced excellent positive signals. the epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. as one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. in the elisa-array assay, this problem exists as well. because of the limitation of available antibodies, this assay could only detect pathogens. in the future, with increasing numbers of suitable antibodies, especially specific antibodies against flavivirus, this elisaarray might be able to test more pathogens and be of greater potential use. to make the assay more amenable to multiple virus detection, the assay protocol was optimized. in addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. heat inactivation was performed by heating the viral cultures at °c for h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. thus, β-propiolactone treatment was chosen as the virus-inactivation method. a conventional elisa is a standard method in many diagnostic laboratories. we compared the elisa-array with a conventional elisa and confirmed that the advantage of the elisa-array was evident with comparable specificity and higher sensitivity than elisa. the time required for the elisa-array is significantly less than for conventional elisa ( h vs. a minimum of h, respectively). furthermore, less igg is required for printing than for coating elisa plates. coating of a single well in microtiter plate requires μl of a μg/ml antibody solution, which is equivalent to ng of igg. for the elisa-array, only nl of a μg/ml antibody solution is required for each spot, which is equivalent to . ng of igg. with the characteristics of ease of use, sensitivity, specificity, and accuracy, the elisa-array assay would be widely accepted for clinical use. direct broad-range detection of alphaviruses in mosquito extracts multiplexed protein measurement: technologies and applications of protein and antibody arrays multiplexed sandwich assays in microarray format detection of adamantane-resistant influenza on a microarray a novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis advances in viral disease diagnostic and molecular epidemicological technologies assessment of some tools for the characterization of the human osteoarthritic cartilage proteome high-throughput microarray-based enzyme-linked immunosorbent assay (elisa) development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis multiplexed sandwich assays in microarray format validating a custom multiplex elisa against individual commercial immunoassays using clinical samples application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer human neutralizing sars-cov specific fab molecules generated by phage display simultaneous multianalyte elisa performed on a microarray platform a multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis c virus infection and typing of whole blood a simple and rapid protein array based method for simultaneous detection of biowarfare agent microarray immunoassay for the detection of grapevine and tree fruit viruses development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna a duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses high diagnostic accuracy of antigen microarray for sensitive detection of hepatitis c virus infection the role of biosensors in the detection of emerging infectious diseases submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the national natural science foundation of china (grant no. ) and national key research special foundation of china (grant no. zx - ). authors' contributions xk: designed the study, performed the laboratory testing, analyzed the test results, and co-wrote and edited the manuscript. h l and yl performed the virus cultures. lf performed laboratory testing. xc, fl, and gc performed real time-pcr. qz and yy organized the overall project and helped edit the manuscript. all of the authors read and approved the final manuscript. state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology, beijing , china the authors declare that they have no competing interests. key: cord- -b shffb authors: shaukat, shahzad; angez, mehar; alam, muhammad masroor; jebbink, maarten f; deijs, martin; canuti, marta; sharif, salmaan; de vries, michel; khurshid, adnan; mahmood, tariq; van der hoek, lia; zaidi, syed sohail zahoor title: identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified vidisca date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: b shffb background: the use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. one sequence independent method, virus discovery based on cdna amplified fragment length polymorphism (vidisca) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. methods: vidisca is normally combined with next generation sequencing, however, we set up a simplified vidisca which can be used in case next generation sequencing is not possible. stool samples of patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. to further characterize the viruses, vidisca-positive samples were amplified and sequenced with gene specific primers. results: simplified vidisca detected seven viruses ( %) and the proportion of eukaryotic viral sequences from each sample ranged from . to . %. human enterovirus ev-b , ev-b , echovirus- and echovirus- , human parechovirus type- , human astrovirus probably a type- / recombinant, and tetnovirus- were identified. phylogenetic analysis based on the vp region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. conclusion: our data support that a simplified vidisca protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients. electronic supplementary material: the online version of this article (doi: . / - x- - ) contains supplementary material, which is available to authorized users. the identification and characterization of viruses in clinical samples is an essential component of community health monitoring systems. a diverse range of conventional and molecular based diagnostic assays are widely used to detect these pathogens. conventional assays like electron microscopy, cell culture and immunological methods have been successfully used for identification of viruses but in many occasions these methods failed to detect the etiological agent in clinical samples due to poor sensitivity and cross reactivity. therefore, molecular assays like polymerase chain reaction (pcr) [ ] , universal primer pcr [ ] [ ] [ ] , pan viral microarray, cdna library immunoscreening [ ] , substitution hybridization [ ] [ ] [ ] have been used for detection of unknown viruses, but these methods were not always proven useful due to increased diversity of viral genome, low viral loads in clinical samples and presence of a variety of organisms in a single specimen. therefore, there is a need to develop improved and economical diagnostic tools to deal with such problems. in recent years the technological innovations involving sequence independent amplification techniques and next generation sequencing (ngs) have boosted virus discovery and these ngs-based techniques are becoming the standard for the discovery of viral pathogens in clinical samples. among these innovations, sequence independent methods have been used efficiently to identify unknown viruses in diagnostic virology. one of these methods, virus discovery based on cdna amplified fragment length polymorphism (vidisca), has proven to be a successful tool for identification of unknown viruses [ ] . this method is capable of detecting both dna and rna viruses without prior knowledge of the viral family, and is based on restriction enzyme digestion. nowadays vidisca is combined with next generation sequencing, but at the time it was developed ( ), it has been successfully used to discover novel viruses from cell culture like human coronavirus nl (hcov-nl ) [ ] , human parechovirus type and [ ] and human parechovirus type variant [ ] . standard vidisca uses a two step pcr amplification protocol with a complex second selective amplification round, and subsequent isolation of pcr fragments which are not in uninfected cultures. this last amplification step is complex and labor intensive, but needed to distinguish background ribosomal rna pcr fragments from the viral pcr products. however, pre-treatment of samples prior to vidisca has significantly been optimized and amplification of ribosomal rna has diminished dramatically [ ] . therefore, a simplified vidisca protocol was developed, which lacks the last amplification round, and evaluated using viruses that were cultured from stool samples of acute flaccid paralysis children, and which had remained unrecognized on both cell culture and enterovirus specific real-time pcr. virus discovery techniques are often a combination of unbiased amplification and high throughput sequencing. however, high throughput sequencing is almost impossible in developing countries like pakistan because its cost is prohibitive. therefore, we used a simple and cheap alternative virus discovery tool, which is a combination of a short virus culture together with a simplified version of vidisca (an overview of the adaptations is shown in additional file : table s ). ten supernatants from cpe-showing cultures of stool from patients with acute flaccid paralysis were tested (patient characteristics are shown in table ). the virus cultures had been examined by the standard diagnostics but remained negative (data not shown). all of the study samples yielded pcr fragments on % metaphor gel ( figure ). of each sample the pcr products which were present in the samples but not in the cell culture control were cut from gel and cloned in e.coli. twelve to cloned products were sanger sequenced. a significant amount of nucleotide sequences showed identity to known viruses in samples ( %). these viral sequences matched with members belonging to different families e.g. picornaviridae, astroviridae ( table ). the proportion of eukaryotic viral sequences in each sample varied and ranged from . % to . %. four human enteroviruses were identified (serotypes ev-b , ev-b , echovirus (e)- and e- ), human parechovirus type (hpev- ), tetnovirus- (tnv- ), and one human astrovirus (several fragments of which some with identity to type (hastv- ) and other fragments -at different locations of the genome -showing identity with human astrovirus type (hastv- ). to further characterize the viruses, the vidisca-positive samples were amplified and sequenced with gene specific primers. for the human enteroviruses and the parechovirus the vp gene was used, and the orf a and orf genes were used for the astrovirus. phylogenetic analyses of the vp gene were performed to investigate the genetic relationships between the human enterovirus strains from this study (pak-nih vs a, vs , vs a and vs ) and the enterovirus serotypes in genbank ( figure ). the nucleotide identity between the study strains and their reference prototypes ranged from . % to . % and within each serotype from . % to . %. the nucleotide sequence identity between pak-nih-vs a and the closest relative echovirus (farina;af ) was only . % and thus this isolate represents a separate genotype, matching most closely with strains from india, china and sweden (genbank accession numbers are shown in figure ). phylogenetic analysis of the vp coding region of hpev strains available in genbank and our study isolate (pak-nih-vs ) showed clustering with hpev type strain (fj ) isolated in amsterdam, the netherlands ( figure ). the strains had % nucleotide identity and % amino acid identity with each other. the orf a and orf sequences of astrovirus pak-nih-vs were aligned and compared with sequences from genbank including those who gave the largest similarity in blast alignments. the genetic analysis in orf a gene (serine protease) showed that the study isolate belongs to human astrovirus type representing . % nucleotide identity and % amino acid identity with hastv isolate dl (jq ). on the other hand, genetic analysis in orf gene (capsid protein) showed that pak-nih-vs shared . % nucleotide and % amino acid similarities with hastv type isolate idh (ab ), a finding which matches with the results from vidisca and we conclude that the astrovirus is a recombinant, with a recombination site between orf a and orf . phylogenetic trees were constructed based on sequences of both genes separately which showed that pak-nih-vs has different grouping patterns in relation to the reference hastv prototypes (figure ), confirming that the isolate is most probably a recombinant ( figure ). the partial nucleotide sequence of pak-nih-vs ( -nucleotides) showed most identity with tetnovirus- strain (hm ) isolated from an afp patient in afghanistan ( figure ). pairwise distance calculation showed . % nucleotide identity with its closest match and therefore pak-nih-vs probably presents a divergent isolate within this group. in recent years, sequence independent pcr approaches have revolutionized identification of unknown and novel viruses either alone or in combination with conventional methods. in this study a simplified version of vidisca was used to detect human enteroviruses, a human parechovirus type , a human astrovirus, and a tetnovirus- in cell culture. our data show that a wide range of distinct viruses that remained unrevealed through routine assays can be identified in a relatively short amount of time with an easy to use method. the vidisca method is based on cdna-aflp and one characteristic of this method is that it uses a double pcr strategy, with in the second round of amplification selective primers (selective pcr-round). these selective primers are extended at the ′ site with one or two nucleotides, and in the selective pcr various combinations of primers are used in order to amplify everything which was amplified in the first pcr. this selective pcr step is a complex and laborious step in vidisca, but was needed previously to distinguish viral pcr fragments from ribosomal rna amplicons. however, an improved purification and reverse transcription step which strongly diminishes ribosomal rna amplification has recently been published, and therefore the laborious selective amplification step might not be needed [ ] . here, we show that in the majority of virus cultures simplified-vidisca can quickly reveal the infecting agent. we previously published that vidisca in its original setting can identify a virus in picornavirus-cultures in > % of the cultures [ ] . a sensitivity of % which we have here is largely comparable, but we must mention here that we did not compare the sensitivity of traditional vidisca and simplified vidisca directly in our study. the possible failure of screening of study samples via routine assays may be due to increased genetic diversity at pcr priming annealing sites [ , ] . seven viruses (ev-b , ev-b , e- and e- , hpev- , hastv- / and tnv- ) were identified which originate from stool samples of afp patients. the clinical significance of these viruses has great impact on public health and constitutes a health risk for the community. all isolated enteroviruses belong to enterovirus b species containing the most frequently isolated serotypes that more commonly cause meningitis, myocarditis and neurological disorders [ ] [ ] [ ] [ ] [ ] . some enteroviruses cause severe and potentially lifethreatening illness and there is currently no antiviral treatment available for enterovirus infection [ ] . phylogenetic clustering of our study isolates reveals that the enteroviruses are related to the circulating strains which have been reported in neighboring countries: india and bangladesh. our analysis also showed that the nucleotide sequence of echovirus isolate (pak-nih vs a) has low nucleotide identity ( . %) with prototype farina strain (af ) that fulfils the criterion for a genotype [ ] and therefore we suggested that it is a separate genotype of e- circulating in the area. importantly, the emergence of new genetic lineages of enteroviruses in the community is an alarming situation for pakistan where there is no enterovirus surveillance system. aside from poliovirus, which is the target pathogen of the polio eradication strategy, the non-polio enterovirus detection in the laboratory is only a "part-outcome" of afp surveillance. in pakistan, despite the significant number of isolated nonpolio enteroviruses, limited information is available with regard to its incidence, diversity and circulation pattern. consequently, it is the right time to prepare for future tasks and to give attention towards non polio viruses causing afp which is an equal cause of concern while we are approaching towards the polio eradication era. similarly, hpevs are known to cause a variety of clinical symptoms similar to enteroviruses like gastroenteritis and occasionally flaccid paralysis and encephalitis [ , ] particularly in infants, and they are considered a major cause of infant mortality worldwide. hpev types , , , and were isolated from stool samples of non polio afp patients < years of age [ ] [ ] [ ] . in this study we identified hpev- in a seven months old paralytic infant having fever. our findings was in agreement with previous studies in which hpev- was isolated from children aged less than three years having transient paralysis and sepsis like illness [ , ] . astrovirus is one of the known causes of acute gastroenteritis in humans, mostly in young children. it has been isolated earlier from stool of an afp patient [ ] . in this study, an astrovirus was isolated from a six months old child. a follow up examination after days indicated the complete recovery of patient from paralysis. phylogenetic analyses of pak-nih-vs based on orf a and orf genes clustered this isolate in two different genotypes; hastv- and hastv- . this genotype discrepancy among phylogenetic analysis of both genes most likely represents a possible recombination event in the evolution of hastvs. recombination is a normal phenomenon among these viruses [ ] [ ] [ ] [ ] . pak-nih-vs is the most divergent strain of the type tetnoviruses. tetnovirus- is an rna virus containing two large orfs encoding structural and non-structural proteins. the non-structural protein of tetnovirus- contains an rna dependent rna polymerase (rdrp) and a cysteine-like protease domain. the genomic organization of tnv- is more closely related to the viruses classified in the family tetraviridae. tetnoviruses are closely related to nodaviruses within the rdrp protein. the host of nodaviruses is fish and arthropods, including insects. tetnovirus- and − have been isolated earlier from [ ] . future studies are needed to identify the origins of these viruses and to clarify their in vitro replication and pathogenic potential in afp children. nowadays sequence independent methods followed by high throughput sequencing [ ] [ ] [ ] are becoming more promising means for detection of novel pathogens. however, highly developed technical skills, involvement of bioinformatics' support, adequate computing resources, advanced data interpretation and high costs are major barriers for their use in developing and resource-limited countries, which are exactly those regions where outbreaks of new pathogens are likely to occur [ , [ ] [ ] [ ] . therefore, we consider the adaptation of vidisca a most appropriate method for resource poor countries and it can be successfully completed in a limited time without technical difficulties. this assay is applicable independently in any laboratory having only pcr and sequencing facilities and can be reproduced easily from the literature. our data support that vidisca may be of great utility for the identification of viruses that escaped conventional diagnostics. furthermore, avoiding the cost, time, labor and use of specialized equipments made it an efficient and powerful diagnostic tool for resource poor countries. finally, our findings also provide confidence to improve and redesign the routine diagnostic assays which might lead to additional detection of previously undiagnosed viruses in clinical samples of patients. a total of ten stool samples from afp children aged less than years were selected from the sample bank of acute flaccid paralysis patients at the virology department, national institute of health, pakistan (table ). all these samples showed a cytopathic effect (cpe) in rhabdomyasarcoma cells (rd) and mouse cells that have receptors for human polioviruses (l b) and found negative for enterovirus by real-time reverse transcription pcr targeting the ′utr of the genome [ ] . cell culture controls each for l b and rd cells were included this study was approved by the internal review board of national institute of health, pakistan. written informed consent was obtained from parents (or guardians) of participating patients. the pretreatment of samples was used prior to vidisca to reduce background nucleic acids. samples were centrifuged ( . g) and supernatant was digested with trubo dnase (ambion). extraction and isolation of nucleic acids was performed according to the protocol of boom et al. [ ] . reverse transcription was performed using random hexameres, designed such that they do not anneal to ribosomal rna as described by endoh et al. [ ] , and klenow polymerase was used for second strand synthesis (new england biolabs). the double stranded dna was subsequently digested and annealed to adaptors as described by de vries et al. [ ] , followed by pcr, only one round of cycles, which is different from the protocol as described [ ] a cookbook version of the method is presented as additional file : (doc. s ). sequences were trimmed from raw sequencing data using sequencher programe version . (gene codes corporation) and analyzed online (http://www.ncbi.nlm.nih.gov/blast). the blastn and blastx algorithms were used to identify sequences with similarity to known viruses in genbank and those sequences identified as viral were further classified into viral families based on the taxonomy of the best hit. all samples with sequence identity to known viruses were characterized with gene specific pcrs to confirm the presence of the pathogen in the samples. the capsid-encoding vp gene of human enteroviruses and human parechovirus type was amplified by using primers / , / [ ] and vp -parechof /vp -parechor [ ] respectively. similarly orf a and orf genes of human astrovirus were amplified by using primers mon /mon and mon /mon [ ] respectively. amplified dna products were visualized on % agarose gel and sequenced with the same primers as used in pcr. phylogenetic analyses were conducted using mega (molecular evolutionary genetic analysis) version . 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characterization of a new type of bovine enterovirus a newly identified bocavirus species in human stool a highly prevalent and genetically diversified picornaviridae genus in south asian children rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by realtime reverse transcription-pcr using degenerate primers and probes containing deoxyinosine residues rapid and simple method for purification of nucleic acids species-independent detection of rna virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription species-specific rt-pcr amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses human parechovirus infections in dutch children and the association between serotype and disease severity mega : molecular evolutionary genetics analysis (mega) software version . a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified vidisca key: cord- -pwps y authors: garrido, jose l; maruo, seijii; takada, kenzo; rosendorff, adam title: ebna c interacts with gadd and counteracts the unfolded protein response date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: pwps y ebna c is an ebv-encoded nuclear protein, essential for proliferation of ebv infected b-lymphocytes. using ebna c amino acids - in a yeast two hybrid screen, we found an interaction with the growth arrest and dna-damage protein, gadd . when both proteins are overexpressed, gadd can interact with ebna c in both nuclear and cytoplasmic compartments. amino acids - of gadd , including the two pp a interaction, and the hsv- icpγ . homology domains, are required for the interaction. furthermore, interaction is lost with a mutant of ebna c (( )dvievid ( )→aviavia), that abolishes ebna c coactivation ability as well as sumo interaction[ ]. in b-cells, gadd , and ebna c are present in a complex with pp a using microcystin sepharose affinity purification, using a lymphoblastoid cell line in which ebna c protein levels are conditional on hydroxytamoxifen, surprisingly, we found that (i) ebna c maintains phosphorylation of eif α at serine , and (ii) protects against er stress induced activation of the unfolded protein response as measured by xbp (u) versus xbp (s) protein expression and n-terminal atf cleavage. in reporter assays, overexpression of gadd enhances ebna c's ability to co-activate ebna activation of the lmp promoter. collectively the data suggest that ebna c interacts with gadd , activating the upstream component of the upr (eif α phosphorylation) while preventing downstream upr events (xbp activation and atf cleavage). epstein-barr virus is a ubiquitous human herpes-virus that causes infectious mononucleosis. it remains latent in bcells following resolution of infection, however, it has the potential to be a serious opportunistic pathogen. expression of ebv latency iii proteins is observed in acute infection, as well as in ebv positive post-transplant, and xlinked lymphoproliferative disease (ptld and xlp) and hiv associated cns lymphoma [ ] . in this pattern of gene expression, nuclear proteins (ebnas , a, b and c), three integral membrane proteins (lmp , lmp a and lmp b) and two non-coding poly-adenylated rnas (ebers and ) are expressed [ , ] . expression of these genes converts b-cells to leukemic lymphoblasts in vivo, and to lymphoblastoid cell lines in vitro. ebna c, is essential for initiation of b-cell growth, as well as ongoing b-cell transformation. recombinant ebv containing a stop codon in the ebna c orf is able to cause b-cell transformation only when transcomplemented for wildtype ebna c either in cis or trans, and lcls immortal-ized by recombinant ebv containing a conditional ebna c gene, undergo growth arrest when ebna c expression is turned off [ ] [ ] [ ] ebna c co-activates transcription with ebna at the viral lmp- promoter, as well as heterologous reporter systems designed to test p function. ebna c amino acids - were found to be essential for co-activation in both reporter systems, and yeast two-hybrid studies established that aa - are sufficient for interaction with both sumo- and with sumo- [ ] we further established that ebna c uses a sumo interaction motif (sim) (aa - ) to interact with sumo- and sumo- , and that co-activation with ebna , is lost with mutations of the sim (eg. m , dvievid →aviavia) that prevent sumo binding, as well as with larger deletions (eg Δ - ) that remove the central portion of the protein including the sim, but leave other structural domains (eg the rbp-j-kappa binding domain) intact [ ] . in an effort to define other transcriptional activators associated with ebna c in sim dependant manner, we performed a yeast two hybrid assay using ebna c aa - as bait, and a splenic b-cell yeast two hybrid library as prey. suprisingly, ebna c was shown to interact robustly with the growth arrest and dna-damage protein (gadd ), an er-associated protein that is up-regulated in response to viral infection as well as er-stress. furthermore, interaction with gadd was lost when we tested a sim mutated form of ebna c for interaction (m , dvievid →aviavia) in this study we sought understand the effects of the interaction between ebna c and gadd on transcriptional co-activation with ebna at, the - /+ lmp- promoter. since gadd is involved in resuming protein synthesis following resolution of er stress, by functioning as a phosphotase subunit towards eif α, we also sought to investigate ebna c effects on the unfolded protein response in ebv infected b-lymphocytes. in this study, we map a region important for ebna c interaction with gadd , and show that gadd can cooperate with ebna c in co-activation of the lmp promoter with ebna , in a manner that depends on ebna c interaction with gadd , but appears independent from gadd binding to pp a. using a cell line (lcl c - ) conditional on tamoxifen for ebna c expression, surprisingly, we find that ebna c expression results in an increase in eif α serine phosphorylation, an early event in both the pkr and unfolded protein responses. paradoxically, ebna c protected against downstream events in the upr, namely the switch from expression of unspliced to spliced xbp isoforms, as well as atf cleavage. ebna c's interaction with gadd may therefore sustain lmp promoter activation in latency iii infection, while preventing stress-induced activation of the upr. plasmids psg -ebna , psg- ebna c - , psg- ebna c - dvievid → aviavia ( m ) and reporter plasmids - /+ lmp- -luc, and pgkb-gal, have been described previously. plasmids pas ebna c aa - was constructed by subcloning the bamhi/sali fragment from pgex- x-ebna c aa - into the bamhi and sali sites of pas- (gift of s. elledge). pas ebna c aa - -m was constructed by subcloning the spei/aatii fragment from psg- ebna c-m into pas- ebna c aa - . psg -flag-gadd - , - , - and - were kindly provided by m brush and s shenolikar, duke university. anti-human gadd (ab ), anti-xbp (ab ), anti-laminin b (ab ), and anti-eif α (ab ) antibodies were purchased from abcam inc. anti-phospho-eif α (ser ) antibody was obtained from cell signaling technology. anti-ebna c was purchased from exalpha biologicals inc. anitbody recognizing only the spliced xbp isoform was obtained from biolegend. anti-atf antibody, recognizing only the kda isoform, was purched from imgenex (san-diego, ca). anti-β-actin (a ) antibody was obtained from sigma-aldrich. yeast two hybrid analysis μg of pas- ebna c aa - and a pact library were co-transformed into . ml of log phase yeast strain ah . yeast were spread on mm synthetic dropout (sd) plates (leu -, trp -, his -, ade -,β-gal) after weeks of growth, blue colonies were selected and replated on sd plates (leu -, trp -, his -+ mm -at). / colonies that grew under these conditions, were selected and transforming plasmids segregated by two cycles of liquid culture amplification and restreaking sd (leu -, trp -, x-β-gal plates), yielding a mixture of blue and white colonies. blue colonies were picked and yeast minipreps performed, followed by transformation of dh α max efficiency cells (life technologies, rockville, md) and miniprep. duplicate cdnas were identified by pcr across the pact mcs, followed by alu digest of pcr products. unique cdnas were then retested by retransformation into ah cells and growth on sd media (leu -, trp -, his -, ade -.) confirmed interactors were selected for parallel retransformation of ah with ebna c - , ebna c aa - or ebna c aa - m , followed by plating on sd media (leu -, trp -, his -, ade -). million bjab or bjab-e c cells were collected and lysed in ml buffer containing . % np- , mm nacl with or without added . % bsa. lysates were incubated for hour or overnight with either protein g sepharose (amersham pharmacia, piscataway, nj) or microcystin sepharose (upstate, lake placid, ny). proteins were resolved by electrophoresis and ebna c and gadd were detected using mouse monoclonal a antibody, and anti-gadd rabbit polyclonal antibody (c- , santa cruz) for ib co-immunoprecipitation experiments, million ib cells were lysed in ripa buffer ( % np- , % sodium deoxycholate, . % sds, . molar nacl, . molar sodium phosphate, ph . ) for minutes. insoluble matrix was pelleted by centrifugation at rpm for minutes, and clarified lysates pre-cleared with protein-g-sepharose. protein-g-sepharose or protein-g sepharose with a antibody were added to lysates which were incubated with rotation at degrees overnight. following ip, proteins were resolved by electrophoresis and gadd detected using c- antibody. bjab cells ( ) were transfected in . ml of rpmi supplemented with % neugem serum with a bio-rad gene pulser at v and μf. each transfection contained ug of - /+ lmp -luc plasmid and ug pgk-b-galactosidase plasmid as a normalization control, and the indicated microgram amounts of psg -ebna , psg -ebna c or psg -gadd plasmids. total plasmid dna was made equal across transfections by the addition of psg vector dna. after transfection, cells were placed in ml of complete medium and incubated at for hours. cells were collected, washed in phosphate buffered saline, lysed in reporter buffer (luciferase assay system, promega, madison, wi) by one freeze thaw cycle, and assayed for luciferase and beta-galactosidase activities (galacto-light; tropix) with an opticomp i luminometer (mgm instruments.) stress assays lcl c - cells were maintained in rpmi suplemented with % fbs in the presence of nm -hydroxytamoxifen ( -ht) (sigma-aldrich), and then transferred to medium containing either ht or dmso for days. six hours prior to harvest, cells were treated with thapsigargin ( . μm) to induce er stress and activate the upr. following treatment cells lysed were prepared using ripa buffer. protein concentration was measured using bio-rad protein asssay. equal amounts of protein ( μg) were separated by sds-page and protein transferred to a pvdf membrane. blots were blocked by incubation for h at room temperature in % bovine serum albumin (bsa) in tbs buffer ( mm tris hcl ph . ; mm) containing . % tween- (tbs-t). the membranes were incubated overnight with the indicated primary antibodies diluted in % bsa in tbs-t. membranes then were washed times for min with tbs-t followed by incu-bation with the appropriate secondary antibodies diluted in % bsa in tbs-t for h at room temperature. finally, membranes were washed times with tbs-t buffer for min and immunoreactivity was detected using ecl system obtained from millipore. yeast two-hybrid screening using ebna c aa - as bait, revealed one of the interactors to be the protein encoded by pact-gadd aa - . interaction was confirmed with a β-galactosidase filter lift assay, after an half hour incubation. previous studies showed that ebna c aa - containing the m ( dddvievid →ddaviavia) and m ( dddvievid →aaavievid) interact very weakly with sumo- and fail to interact with sumo- [ ] . to test whether interaction between ebna c and gadd also depends on the sumo interaction motif, the m mutation was constructed in the context of ebna c aa - , and tested against gadd aa - (table ) . while wild type ebna c aa - interacted with gadd , interaction was lost when the m mutant was tested. as expected, wild type ebna c aa - but not the m mutant, interacted with sumo- [ ] . therefore ebna c aa - interaction with gadd depends on the presence of an intact sumo interaction motif, a domain of ebna c also required for transcriptional co-activation with ebna . in order to test whether full length ebna c interacts with gadd , the gal dna binding domain was fused n-terminal to the entire ebna c orf (aa - ), cloned into y h vector pas- , and tested against pact-gadd aa - . as a positive control a cdna encoding full length rbp-j-kappa, cloned into pact, was tested for interaction with ebna c under the same conditions. full length ebna c interacted with gadd aa - , as focused yeast two-hybrid assays testing the interaction of full-length ebna c, ebna c aa - yeast strain ah were transformed in parallel with pas -ebna c ( - ), pas-wt ebna c (aa - ) or with the sim (m ) mutant (aa - , dvievid → aviavia ) and tested for interaction with pact-gadd aa - . colonies were sampled using a filter lift assay and interaction confirmed using a lacz assay. results of a hour lacz assay, scored semi-quantitatively, are shown. well as with rbp-j-kappa, and lacz conversion occurred within minutes for both interactions. these data indicate that ebna c interacts with gadd aa - , and that the interaction is comparable in affinity to the interaction between ebna c and rbp-jκ. furthermore, ebna c aa - interaction with gadd aa - depends on the sumo interaction motif. these data also suggest that gadd may, in part, be a mediator of ebna c co-activation function. to microcystins are cyclic heptapeptides synthesized by bluegreen algae that covalently couple to the catalytic subunits of protein phosphotases pp and pp a [ ] . although not completely specific to pp a, taken together, the data indicate that both ebna c and gadd reside in protein phosphotase complexes in b cells. since gadd interaction with ebna c depends on a motif that is also required for transcriptional co-activation by ebna c reporter assays in the burkitt's lymphoma cell line, bjab were used to assess gadd affects on ebna c co-activation. the reporter chosen was - /+ of the lmp- promoter fused to luciferase, which contains two rbp-j-kappa binding sites, and is reliably activated by ebna and further activated by ebna c [ ] . this region gadd aa - are required for ebna c association of the lmp promoter also includes an er stress response element (erse) located at position - relative to the transcriptional start site, recently shown to be important for lmp promoter activation by er stress inducing chemicals (eg brefeldin a, tunicamycin) or by overexpression of the er-stress transactivator, xbp [ ] . lmp- promoter activity levels observed with expression of ebna alone was normalized to , (figure a, lane ) . co-transfection of μg ebna c expression plasmid increased reporter activity levels to over fold that observed with ebna alone (lane ) while co-expression of ebna c and full length gadd resulted in a further . fold (from . to . ) increase in promoter activity levels (lane ). full length gadd (aa - , lane ) and gadd - (lane ) were equally effective in cooperating with ebna c, while full length gadd had no effect on basal lmp reporter activity or ebna activity in the absence of ebna c (data not shown). gadd effects on lmp promoter activity depended on both ebna and ebna c expression. in order to test whether the ability of gadd to synergize with ebna c in the co-activation of ebna at the lmp promoter, depended on gadd binding to pp a, point mutants known to affect pp a activity ( kvrf →kara) or association ( - Δrara) were tested in the same reporter assay (figure a). a - . fold potentiation of ebna c promoter activity was again observed with both the kvrf ( . vs . for wt gadd ) as well as Δrara ( . vs . for wt gadd ) mutants (lane versus lanes , and ). these data suggest that enhancement of ebna c activity by gadd does not depend on association with pp a. when a smaller quantities ( ng) of ebna c expression plasmid were used in the same experiment, ebna c expression increased lmp- promoter activity levels sub maximally to . fold relative to ebna alone (figure b , lane ), and this was enhanced approximately -fold by the co-transfection of μg of plasmid expressing gadd (figure b, lane ) . gadd was able to potentiate ebna c transcriptional co-activation in a dose dependant manner (lanes - ). by contrast, a dominant negative effect on ebna c activity was observed with overexpression of a gadd truncation mutant that cannot bind ebna c (gadd aa - , figure b, lanes - ). ebna c interaction with gadd might effect eif α phosphorylation, because gadd recruits pp a to the er where the gadd /pp a holoenzyme dephosphorylates eif a at serine . in order to determine ebna c effects on eif α activation, we used a cell line (lcl- - ) in which ebna c expression is controlled by hydroxytamoxifen. aliquots of these cells were maintained in media containing hydroxytamoxifen and then transferred to media with (+ht) or without (-ht) hydroxytamoxifen, for - days. lysates were prepared and western blotted for the indicated proteins (figure a ) as expected with this cell line, withdrawal of hydroxytamoxifen resulted in slowed cell growth over a period of - days, accompanied by loss of ebna c-ht protein expression. while total eif α levels were comparable in cells grown in the presence of absence of tamoxifen, a significant difference in serine phosphorylation was observed, with little effect on total eif α levels ( figure a ). figure ebna c interacts with gadd by co-ip and co-purifies with gadd by microcystin pulldown. a) ib cells ( million for each treatment) were collected, lysed in isotonic . % np- buffer, and immune precipitations (ip) performed with either protein g alone (pg) or protein g with the addition of anti-ebna c (a ) sera (pg/anti-ebna c) proteins were western blotted for gadd (sc-h ), following ip. b) burkitt's lymphoma bjab cells or bjab-ebna c cells ( million for each treatment) were collected, lysed in isotonic . % np- buffer, containing additional . % bsa, and incubated with the indicated sepharose beads conjugated to either protein g or microcystin lr. lysates and beads were rotated for hr at degrees, extensively washed with pbs, and affinity purified proteins western blotted for the presence of ebna c (e c) and gadd . tion of er stress during the upr [ ] . thapsigargin induces er stress by inactivating the er ca + atpase resulting in depletion of ca + from the luminal er, and dysfunction of ca dependant er proteins such as calnexin. in order to determine whether ebna c affects the upr in lcls, lcl c - was maintained in the presence or absence of hydroxytamoxifen as in a. four hours prior to harvest, cells were incubated in either dmso alone ( collectively these data strongly implicate ebna c in preventing lcls from undergoing upr signaling, following er stress. in this study, a yeast two-hybrid assay was conducted to reveal further ebna c interacting proteins. in particular our primary intention was to find novel proteins that might give clues as to the mechanism of ebna c transcriptional co-activation with ebna . surprisingly, the two-hybrid assay failed to reveal convincing interactions with bona-fide nuclear transcription factors. rather an interaction was discovered between ebna c and the translation control protein, gadd , and robustly confirmed in human cells by immune precipitation. since mutations that affect sumo- and sumo- binding also affect the ability of ebna c to bind to gadd , as well as ebna c's ability to co-activate transcription with ebna , a genetic and biophysical link has been established between ebna c sumo and gadd binding and ebna c transcriptional activity. further experiments clearly indicated that full length gadd , including aa - which were required for ebna c interaction, have a synergistic effect with ebna c in co-activating transcription with ebna at the lmp promoter, while having no effect on ebna driven reporter activity in the absence of ebna c. indeed, a mutant of gadd , fl-gadd aa - , acted as a dominant negative in reporter assays. it is known that the chromatin remodeling swi-snf protein, ini- also associates with the c-terminus of gadd explaining the positive effect on transcription. at higher concentrations ( and micrograms), gadd - functioned as a dominant negative, reversing ebna c coactivation. since this mutant does not associate with ebna c, and is therefore likely not targeted to sites of ebna c activity on chromatin, the effect is likely due to swamping of the cell with gadd protein, and sequestration of a positively acting factor such as the histone-acetyl transferase cbp, which is known to be required for full ebna activity at the lmp promoter. ebna c, a nuclear protein, would not be expected to interact with gadd , a cytosolic and er-associated protein. however, ebna c might have the opportunity to associate transiently with gadd at the er following translation, during dismantling of the nuclear lamina during mitosis, or during lysosomal or proteasome or mediated degradation. indeed ebna c and ebna a have been shown to associate with cytosolic proteasome subunits [ ] furthermore, while genetic studies have proven a role for ebna c in maintaining growth of ebv trans-a: ebna c effects on phosphorylation the translational con-trol protein eif α figure a: ebna c effects on phosphorylation the translational control protein eif α. lcl - was maintained in the presence (+ht) or absence (-ht) of hydroxytamoxifen for days. shown are western blots from whole-cell lysates against ebna c (top row), serine phosphorylated eif α (second row), total eif α (third row) and β-actin loading control (fourth row). by day , in the absence of ht, lcl - cells had stopped dividing [ ] . b: ebna c protects lcls from activating the unfolded protein response. as in figure a except that cells were also treated with either dmso "c" or . μm thapsigargin "tg", for the hrs preceding harvest. xbp (s) and xbp (u) were individually detected using isoform specific antibodies. an atf antibody that detects only full length, uncleaved atf ( kda) was used (imgenex, san diego, ca). * formed b-lymphocytes, and ebna c biochemically fractionates to the nuclear compartment, it is unknown at present whether nuclear localization of ebna c per se, is required for ebv immortalization. how might gadd be important for ebna c transcriptional activity? one hypothesis, supported in the current study, is that ebna c negatively regulates gadd , decreasing the gadd -dependant pp a recruitment to eif α, and consequently, increasing eif α phosphorylation. since nuclear translocation of transcription factors involved in the pkr and upr responses (such as atf , atf and xbp ) usually occur downstream of eif α serine phosphorylation, a negative effect on gadd function might be expected result in higher levels or greater activity of these transcription factors in the nucleus. indeed there are numerous atf /xbp binding sites in the native lmp promoter as well as the - /+ construct used in this study, and expression of lmp has recently been shown to exert a feed forward effect by increasing eif α serine phosphorylation, and downstream activation of atf [ ] . at least two er stress inducible elements have been described. these are controlled by er stress induced transcription factors as well as xbp . the first at position - relative to the transcriptional start site of the lmp gene, appears active in lcls, is responsible for both ebna dependant and ebna independent activation of the lmp promoter in reporter assays, and is regulated by binding of atf transcription factors [ ] [ ] [ ] . the other erse (position - ) is active in npc but not b-lymphocyte cell lines and is strictly required for lmp promoter activation by xbp overexpression or er stress inducing chemicals such as brefeldin a [ ] our initial hypothesis, therefore, was that ebna c potentiated upr signaling, via an interaction with gadd , increasing atf / and/or xbp activity, to coactivate lmp reporter activity with ebna . consistent with a negative effect on gadd activity, ebna c expression was indeed associated with increased eif α serine phosphorylation. surprisingly, however, we found that ebna c expression was associated with lower levels of active xbp (s), higher levels of inactive xbp(u), and increased levels of uncleaved (inactive) atf protein in lymphoblastoid cells. assuming that atf and xbp transcription factors exert a positive effect on the lmp promoter in lcls, gadd is therefore unlikely to potentiate ebna c transcriptional effects by positively regulating atf or xbp activity. in our hands, ebna c's protective effects in lcls appear similar to effects of the chemical salubrinal on lcls (data not shown), which has the effect of maintaining low levels of serine phosphorylated eif α while preventing downstream events of the upr such as the switch from xbp(u) to xbp (s) protein expression, and activation by proteolytic cleavage of atf in the golgi. salubrinal has been shown to protect neurons against er-stress induced apoptosis, and ebna c may perform a similar role in lcls [ ] . interestingly, salubrinal also effects the efficiency of hsv- lytic replication, possibly through counteracting the effect of icpγ , on maintaining eif α dephosphorylation [ ] . during the course of coronavirus infection, eif a is also phosphorylated, resulting in a decrease in translation of host mrnas, presumably favoring viral mrna translation [ ] . prolonged er stress can result in apoptosis via ask binding to ire , jnk activation, and phosphorylation of bcl or chop mediated downregulation of bcl levels [ ] it therefore it seems plausible that ebna c is anti-apoptotic under conditions of prolonged er stress such as might occur during initial ebv infection of resting b lymphocytes. xbp , in concert with protein kinase d, can activate lytic ebv promoters such as the bzlf (zta) promoter [ ] consistent with tight control of type iii latency, we detect minimal xbp (s) protein under normal lcl growth conditions, slightly increased xbp (s) protein upon ebna c withdrawal, and robust xbp (s) expression under conditions of ebna c withdrawal in the face of chemical induced er stress. we have also demonstrated that lcls lytic promoters such as bzlf , bmrf and brlf are activated by thapsigargin (manuscript in preparation). it seems likely, therefore, that one function of ebna c would be to maintain type iii latency restriction, via shutoff of the upr. physiologically, ebv infected b-lymphocytes may be forced into lytic reactivation in the oropharyngeal mucosa, possibly undergoing er stress secondary to bcr activation. ebna c effects on transcriptional co-activation with ebna may be separable from effects on the upr, because the gadd specific enhancement of ebna c transcriptional effects is maintained with overexpression of a gadd mutant deficient in pp a binding. however these results should be interpreted with caution. while the Δrara mutation eliminates pp a binding, protein phosphotases are notoriously unstable in cells. therefore apparent loss of pp a interaction observed with this mutant, may in fact merely represent decreased association, and it is more likely that these mutations reduce pp a phosphotase function and interaction rather than abolish it completely [ ] . while viral infection activates pkr via dsrna or interferon, perk can be activated by er stress. both pathways of kinase activation occur during viral infection. both pkr and perk can phosphorylate eif α at serine , arresting cellular protein synthesis, as well as the synthesis of latent or lytic viral proteins. gadd recruits pp a to the endoplasmic reticulum where it dephosphorylates eif α, and is therefore essential in maintaining protein synthesis during viral infection [ , ] herpesviruses have evolved mechanisms to counteract both pkr activation via us expression, and eif α phosphorylation through icpγ . expression. specifically, the hsv- encoded icpγ . protein accelerates and maintains the dephosphorylated state of eif α to ensure ongoing translation of viral mrnas [ ] . in this study, ebna c expression had the opposite effect, increasing rather than decreasing eif a phosphorylation, while paradoxically preventing the activation of downstream upr. ebna c may therefore use the upr pathway to alter the levels, activity, or network association, of numerous transcription factors in infected b cells, thereby modulating transcription of essential viral (eg lmp ) and cellular genes, while preventing upr signaling, lytic reactivation, and downstream er stress induced apoptosis. these data presented in this study provide motivation for further studies of the effects of ebna c and other latency iii proteins on the unfolded protein and pkr responses in ebv infection. ebna c coactivation with ebna requires a sumo homology domain epstein-barr virus-induced posttransplant lymphoproliferative disorders. asts/astp ebv-ptld task force and the mayo clinic organized international consensus development meeting epstein-barr virus latent and replicative gene expression in post-transplant lymphoproliferative disorders and aids-related non-hodgkin's lymphomas. french study group of pathology for hiv-associated tumors the expression of epstein-barr virus latent proteins is related to the pathological features of post-transplant lymphoproliferative disorders epstein-barr virus nuclear proteins ebna- a and ebna c are essential for b-lymphocyte growth transformation epstein-barr virus nuclear protein ebna c is required for cell cycle progression and growth maintenance of lymphoblastoid cells epstein-barr virus nuclear protein ebna c residues critical for maintaining lymphoblastoid cell growth epstein-barr virus nuclear antigen c putative repression domain mediates coactivation of the lmp promoter with ebna proteomic characterization of protein phosphatase complexes of the mammalian nucleus endoplasmic reticulum stress triggers xbp -mediated up-regulation of an ebv oncoprotein in nasopharyngeal carcinoma xbp regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response epstein-barr virus ebna proteins bind to the c /alpha subunit of the s proteasome and are degraded by s proteasomes in vitro, but are very stable in latently infected b cells the lmp oncogene of ebv activates perk and the unfolded protein response to drive its own synthesis an atf/cre element mediates both ebna -dependent and ebna -independent activation of the epstein-barr virus lmp gene promoter response to camp levels of the epstein-barr virus ebna -inducible lmp oncogene and ebna inhibition of a pp -like activity a selective inhibitor of eif alpha dephosphorylation protects cells from er stress icp . -dependent and -independent activities of salubrinal in herpes simplex virus- infected cells coronavirus infection modulates the unfolded protein response and mediates sustained translational repression mediators of endoplasmic reticulum stress-induced apoptosis x-boxbinding protein activates lytic epstein-barr virus gene expression in combination with protein kinase d growth arrest and dna damage-inducible protein gadd targets protein phosphatase alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor . mcb feedback inhibition of the unfolded protein response by gadd -mediated dephosphorylation of eif alpha the gamma( ) . protein of herpes simplex virus complexes with protein phosphatase alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor and preclude the shutoff of protein synthesis by double-stranded rna-activated protein kinase the authors wish to thank alan wells for valuable discussions. matt brush and ellen mcfarland provided much needed reagents. this study was supported by a grant from the leukemia and lymphoma society (lls - , to a.r.), and by university of pittsburgh startup and the university of pittsburgh's competitive medical research fund (cmrf) to a.r. the authors declare that they have no competing interests. key: cord- -x c ogo authors: li, jingjing; yang, yongbo; dong, yanming; li, yongshu; huang, yu; yi, qianhui; liu, kaiyu; li, yi title: key elements of the human bocavirus type (hbov ) promoter and its trans-activation by ns protein date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: x c ogo background: human bocavirus (hbov), a parvovirus, is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in humans. all mrnas of hbov are transcribed from a single promoter. methods: in this study, we constructed egfp and luciferase reporter gene vectors under the control of the hbov full promoter (nt – ) and its mutated variants, respectively. fluorescence microscopy was used to observe expression activities of the egfp. dual-luciferase reporter vectors were employed in order to evaluate critical promoter elements and the effect of ns protein on promoter activity. results: the hbov promoter activity was about . -fold and . -fold higher than that of the cmv promoter in t and hela cells, respectively. the putative transcription factor binding region of the promoter was identified to be located between nt and nt . mutations introduced in the caat box of the hbov promoter reduced promoter activity by %, whereas nucleotide substitutions in the tata box had no effect on promoter activity. the hbov promoter activities in t and hela cells, in the presence of ns protein, were - to . -fold higher than those in the absence of ns protein. conclusion: the hbov promoter was highly active in t and hela cell lines, and the sequence from nt to nt was critical for the activity of hbov promoter. the caat box, in contrast to the tata-box, was important for optimum promoter activity. in addition, the transcriptional activity of this promoter could be trans-activated by the viral nonstructural protein ns in these cells. human bocavirus (hbov ) was first discovered in respiratory samples from children [ ] and classified in the genus bocavirus (subfamily, parvovirinae; family, parvoviridae) [ ] with other members, including bovine parvovirus (bpv), minute virus of canines (mvc), gorilla bocavirus (gbov) and porcine bocavirus (pbov) [ ] [ ] [ ] [ ] . three different genotype members of human bocavirus, hbov , and , have been discovered subsequently in fecal specimens. the hbov virus is the most frequently identified pathogen worldwide with infection rates ranging from % to % in respiratory specimens from children under years old with acute respiratory illnesses, often showing a high presence of co-infection with other respiratory viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one of the most frequently observed clinical symptoms in hbov -infected patients is acute wheezing [ , ] . however, vicenti et al. [ ] reported the detection of hbov in stool samples from children under years old with acute gastroenteritis without respiratory tract disease and mitui et al. [ ] detected hbov in the cerebrospinal fluid of children with encephalitis. therefore, the role of hbov in disease requires further investigation. hbov is a linear, single-stranded dna virus with a genome of about nt. recently, huang et al. [ ] obtained the sequence of a full-length hbov genome (including both termini) and demonstrated that this hbov plasmid replicated and produced viruses in human embryonic kidney cells. based on hbov dna sequences detected in hbov -positive clinical samples, lüsebrink et al. [ ] proposed that hbov may have a different replication mechanism than parvoviruses in general. in the hbov genome, there are two major open reading frames (orfs) encoding non-structural protein ns , and capsid proteins vp and overlapping vp , respectively. an additional mid-orf is thought to encode the non-structural protein np with an unknown function. all mrnas of hbov are transcribed from a single promoter located near the '-terminus of the viral genome [ ] , similar to that of the closely related bpv and mvc. recently, a comprehensive transcription profile of hbov by transfecting a replicative chimeric hbov genome in cells and a non-replicative genome in human lung epithelial a cells was generated and the expression profiles of both the structural and non-structural proteins of hbov were studied in detail [ ] . the non-structural ns protein of parvovirus is central to the viral life cycle and plays multiple roles, mediating viral genome transcription, replication, and packaging. the ns protein of parvovirus b is essential for the activation of the b p promoter [ ] and has a positive feedback effect on the activity of the p promoter [ ] . raab et al. [ ] demonstrated that b -ns protein interacted with the p promoter by direct dna binding to cellular transcription factors sp /sp . the p promoter of the autonomous parvovirus minute virus of mice (mvmp) is strongly trans-activated by the nonstructural protein ns , which interacts with the cellular transcription factors sp , tfiia(α/β) and tbp in vitro [ ] [ ] [ ] [ ] [ ] . the transcripts encoding the ns of hbov which are either spliced or unspliced resulted in respectively a large nonstructural protein ns of approximately kda comparable to the ns of mvc and bpv , and a relatively small nonstructural protein ns - of approximately kda [ ] . however, the functions of hbov -ns protein have not been characterized yet. the whole promoter region (nt - ) was amplified from the pwhl- vector by pcr and verified by sequencing. the promoter sequence was then analyzed for the presence of dna elements known to interact with cellular transcription factors using a transcription factor binding site profile database tfsearch (on-line at http://www.cbrc.jp/research/db/tfsearch.html) (figure ). the analysis of promoter regions showed that a core region of the promoter, from nt to nt , is present, i.e. nucleotides away from the initiation codon of ns . the transcription start site for ns was identified at nt by ' race (data not shown), bp upstream from the atg translation start site of ns protein, which is similar to dijkman's data [ ] . the tata-like sequence was located bp upstream of the transcription initiator sequence (ins), and the caat box was identified at nt , and most potential cellular transcription factor binding sites were located from nt to nt . the pgl -bov-egfp vector containing nt - was transfected into t, a , hela and wi- cell lines to assay the activity of the promoter of hbov in these mammalian cells. in parallel, plasmid pegfp-n containing the egfp gene under the control of the cmv promoter was used as a control. the egfp expression was then observed by fluorescence microscopy at h post-transfection. green fluorescence was detected in all cells transfected with the vector containing the egfp gene under the control of the hbov promoter ( figure ). compared to control transfections with pgl -pcmv, fluorescence was slightly stronger in cells transfected with pgl -bov-egfp. because of variable efficiency of transfection in diverse cell types using lipofectamine reagent, numbers of the four cell lines positive for egfp were different. no nonspecific green fluorescence was found in the control cells. figure map of the hbov promoter with potential binding sites for transcription factors indicated (boxes) and essential elements: tata-box, caat-box and ins. the transcription start site was indicated with bold letter in the ins box. cdxa, caudal-type homeobox protein; c-ets, cellular e twenty six domain transcription factor; creb, camp response element binding; nf-y, ccaat box-binding factor; aml-la, acute myeloid leukemia gene binding region. the prl-tk vector was used as an internal control reporter to correct for possible variable transfection efficiencies of the four cell lines. these cell lines were cotransfected with the prl-tk and pgl -( - ) in order to determine relative activities of the hbov promoter in t, wi- , a , and hela cells. for comparison, the pgl -pcmv construct and the pgl -basic plasmid, without any promoter, were used as positive control and negative control, respectively. the results showed that the hbov promoter exhibited much higher activity in these four cell lines compared to the promoter-less control vector pgl -basic ( figure ). hbov and the cmv promoters revealed almost the same activity in wi- and a cells; however, promoter activity of hbov was about . -fold and . -fold higher than that of cmv in t and hela cells, respectively. these data indicated that the hbov promoter was highly active in human cells. several constructs were created by truncating the '-or '-terminus sequences of the the promoter p (nt - ) and then fused in frame with a luciferase gene to determine the minimal region required for high transcriptional activity of hbov promoter ( figure ). after transfection of hela cells with these constructs, luciferase activity was measured as described in the methods section. results indicated that the full promoter p and some '-terminus truncated promoters, including constructs p , p and p , showed almost the same activity, suggesting that the sequences from nt - were not critical for the promoter activity ( figure ). when the sequences of the '-terminus were further deleted to reach the position at nt (p ) and nt (p ), promoter activity decreased by % and %, respectively, compared to the full promoter p . these results suggested that the putative transcription factor binding region, located from nt to nt , may be important for transcriptional activity. the core promoter region from nt to nt was suggested by promoter prediction software analysis [ ] . however, the construct p , although containing the purported promoter ins and tata box key elements, showed activity nearly at background level. when bp sequences upstream of the core promoter remained to yield the construct p , which contained the caat box, the promoter activity was increased almost -fold compared to the p construct. these data suggested that the additional upstream sequences of the core promoter appeared to be required for the hbov promoter activity. two mutant promoter constructs, pgl -tatamut and pgl -caatmut, were created as described in the methods section to measure the contribution of the tata box and caat box to promoter activity. as illustrated in figure , mutations introduced in the caat box of pgl -( - ) reduced the promoter activity by %, whereas substitutions in the tata box had no effect on promoter activity. we next tested the effect of two transcription factor binding sites, creb and cdxa, which were located in the sequence of nt - , on the activity of hbov promoter. as shown in figure , the mutant constructs, pgl -crebmut and pgl -cdxamut, showed no loss in promoter activity. the ns protein of the parvoviruses exhibits multiple functions, such as transcription factor activities. to examine whether the nonstructural protein ns of hbov is able to modulate hbov promoter activity, the p construct was co-transfected with pgl -pcmv-ns construct into t and hela cells, and the expression of ns protein was confirmed by western blotting using an ns -specific antibody ( figure b ). at h post-transfection, transcriptional activities were determined in three independent tests using luciferase assays. here, the activity of the hbov promoter in cells co-transfected with p and pgl -pcmv-ns mut vector (no ns protein expression) was used as a control. as shown in figure a , the hbov promoter activities in the two cell lines in the presence of ns protein were -to . -fold higher than those in the absence of ns protein. these data showed that the ns protein of hbov trans-activated the viral promoter. hbov is a newly identified pathogen associated with human respiratory tract illnesses. in the present study, of nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections ( . %) were positive for hbov (data not shown). next, we constructed a nearly full-length genome clone without itr structures at the termini, which resembled genotype hbov , as established by dna sequence alignment. the region of the full promoter from nt to , the sole promoter in the viral genome [ , ] , including the translation initiation codon of the ns gene, was amplified by pcr using plasmid pwhl- as a template and cloned into the pbluescript ii vector. bioinformatic analysis indicated that the essential elements of the promoter, including tata-box, caat-box, and ins were present in this region. the full promoter construct was transfected into four human cell lines to examine whether the unique viral promoter is active in mammalian cell lines. the results showed that the promoter was highly active not only in all tested human cell lines, but also in other mammalian cells such as porcine and rabbit cells. furthermore, in t and hela cells, the activity of the hbov promoter even exceeded that of the cmv promoter which is well known to be a highly active in eukaryotic cells. however, the hbov promoter was not functional in the insect sf cell line (data not shown). in our recent study, ns transcripts from the left-hand of the viral genome were detected in pwhl- transfected t cells and other mammalian cells including hela, a , wi- cell lines (data not shown), suggesting that this promoter is unique and active in most mammalian cells. the hbov promoter may become an attractive choice when a strong promoter is needed in a variety of vertebrate cells. no viral dna replication was detected by the southern-blot method (data not shown) in cells transfected with the pwhl- plasmid implicating that itrs were essential for viral dna replication during infection. promoters with different lengths were constructed by truncating its sequence at the '-or '-terminus, or at both ends, to determine the minimal sequence required for maximal activity. luciferase assays showed that the region of nt - at the '-terminus did not contribute to the full promoter activity. the core promoter (p ) including ins and tata box exhibited low activity and a mutant of the tata motif had no effect on the promoter activity. these results indicated that the tata box in the hbov promoter is not required for promoter activity. however, upon addition of upstream sequence to include the regulatory element caat box (p ), promoter activity increased. mutations in the caat box had also an effect on the promoter activity. the promoter activity diminished dramatically when the sequences from nt to nt were deleted, demonstrating that it contained key transcription regulatory elements. however, potential creb and cdxa binding sites were shown, by mutant assays, not to contribute to promoter activity. ns , a major parvovirus non-structural protein is a nuclear phosphoprotein involved in essential stages of the viral life cycle. the ns of human parvovirus b has been shown to trans-activate the p promoter [ ] . chen et al. reported that the ns protein of hbov was also localized in the nucleus [ ] , however, the role of hbov ns protein has not been characterized. we therefore examined the effect of the ns protein on the hbov promoter and found that this protein activated in trans the activity of the promoter in t and hela cells. it is possible that the ns protein functions through binding to the promoter region or, indirectly, by interacting with host transcription factors. deletion of the dna elements, such as an atf/creb consensus site, of the p promoter in b , led to a great reduction of trans-activation by ns to only % of that of the wild-type promoter [ ] . a sequence element similar to the creb site is also present within the hbov promoter region: '-tgacgtat- ' (nt - ), which is proximal to the tata box and may be a putative site for binding by the ns protein ( figure ). in summary, this work provides a framework for further characterization of the promoter and study of the mechanism of transcription and expression of the viral genome. further investigation is needed to elucidate trans-activation mechanisms by viral nonstructural proteins and cellular transcription factors. hbov is a parvovirus associated with respiratory disease in humans. the hbov promoter was active with different strengths in all tested mammalian cell lines transfected with constructs of egfp and luciferase report gene under the control of the hbov promoter. the activity of this promoter is higher than that of cmv promoter in t and hela cells. the regulatory element caat box sustained an enhanced activity of the hbov core promoter. moreover, the promoter was trans-activated by ns protein, suggesting that ns protein plays an important role in hbov transcriptional regulation. a large internal fragment containing nts of the hbov genome was amplified from dna extracted from nasopharyngeal aspirate samples with the specific primers based on the published hbov sequence (genbank accession number id: dq ) and cloned into sali-xbai sites of the pbluescript skii vector to generate the pwhl- (genbank accession (figure ) . the substitution mutant pgl -(crebmut) was made by pcr-based mutagenesis from pgl -( - ), whereby the sequence tgacg motif at nt was mutated to tcaat. similarly, in the mutant pgl -(caatmut), the initiator sequence at nt was mutated from ccaat to ttaat; in the mutant pgl -(cdxamut), the initiator sequence at nt was mutated from tatt to gagg; and in the mutant pgl -(tatamut), the initiator sequence at nt was mutated from tatat to gagag ( figure ). pgl -bov-egfp and pgl -pcmv-ns plasmids were constructed by replacing the luciferase gene with the gfp gene of the pgl -( - ) and the luciferase gene with the hbov ns gene (nt - ) of pgl -pcmv vector, respectively. the ns mutant plasmid, designated pgl -pcmv-ns mut, was constructed by mutating the start codon atg to taa. all constructs were verified by sequencing. prl-tk served as an internal control in the luciferase reporter assay. human embryonic kidney cells ( t), human cervical carcinoma cells (hela), human lung epithelial cells (a ) and sv -immortalized human lung fibroblasts (wi- ) were maintained in dulbecco's modified eagle's medium (dmem) with % fetal bovine serum (fbs) and antibiotics in % co at °c. the cells grown in -well plates at × cells per well were transfected with μg of plasmid; the lipofectamine and plus reagents (invitrogen, grand island, ny, usa) were used following the manufacturers' instructions. for cotransfection, t and hela cells were transfected with recombinant plasmids in -well plates at × cells per well, respectively. for detection of hbov promoter activity, mammalian cell lines including t, a , hela and wi- were transfected with the pgl -bov-egfp construct and the pegfp-n control vector in -well plates. the vector pgl -egfp, which contains the egfp gene without any promoter, was used as a negative control. after h, cells were observed by fluorescence microscopy and pictures were acquired by nis-elements f . software. to compare the activity between hbov promoter and cmv promoter, t, a , hela and wi- cells, cultivated in -well plates, were transfected with ng of pgl -( - ) and pgl -pcmv, respectively. for hbov promoter strength analysis, hela cells cultivated in -well plates were respectively transfected with ng of the promoter mutated constructs. to determine whether the hbov promoter can be regulated by the ns protein, t and hela cells were co-transfected with ng of pgl -( - ) and ng of pgl -pcmv-ns , respectively. cells co-transfected with pgl -( - ) and pgl -pcmv-ns mut were used as a control. as an internal control, production of a second type of luciferase derived from renilla reniformis was achieved by co-transfecting the cells with plasmid prl-tk ( ng) together with the promoterluciferase constructs. at h post-transfection, the cells were harvested and lysed with lysis buffer (promega). the assays for the photinus and renilla luciferase activities were performed sequentially according to the dual-luciferase assay kit manual (promega). all tests were performed in triplicate. to detect the ns expression in cells co-transfected with pgl -( - ) and pgl -pcmv-ns by western blots, t and hela cells were transfected with the two constructs as stated above. at h posttransfection, cells were lysed with ripa buffer containing protease inhibitors (pmsf). total cellular proteins were separated by % sds-page and transferred onto nitrocellulose membranes. the membrane was blocked with tbs containing % skimmed milk for h at room temperature and then incubated for h with the polyclonal anti-ns mouse antibody as primary antibody at a : dilution. finally, the membrane was washed with blocking buffer and then incubated with peroxidaseconjugated goat anti-mouse immunoglobulin g (promoter biotechnology, china) as a secondary antibody for h at room temperature. the bands were visualized by -amino- -ethylcarbazole (aec). cells co-transfected with pgl -( - ) and pgl -pcmv-ns mut were used as a control. the mouse anti-ns polyclonal antiserum was prepared by our laboratory. the mice were obtained from the facility in wuhan institute of virology and the study was approved by the institutional animal experiment commission in accordance with the chinese regulations on animal experimentation. cloning of a human parvovirus by molecular screening of respiratory tract samples structure and organization of the viral genome. london: parvoviruses hodder arnold complete nucleotide sequence and genome organization of bovine parvovirus the canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus identification and characterization of a new bocavirus species in gorillas identification and nearly full-length genome characterization of novel porcine bocaviruses frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus infection human bocavirus in french children detection of human bocavirus in japanese children with lower respiratory tract infections bocavirus infection in hospitalized children human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus quantification of human bocavirus in lower respiratory tract infections in china isolation of human bocavirus from swiss infants with respiratory infections human bocavirus infections in hospitalized children and adults human bocavirus respiratory infections in children deubel v: correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection human bocavirus as the cause of a life-threatening infection human bocavirus (hbov) in children with respiratory tract infection by enzyme linked immunosorbent assay (elisa) and qualitative polymerase chain reaction (pcr) human bocavirus and acute wheezing in children high viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection human bocavirus, a respiratory and enteric virus detection of human bocavirus in the cerebrospinal fluid of children with encephalitis establishment of a reverse genetics system for studying human bocavirus in human airway epithelia detection of head-to-tail dna sequences of human bocavirus in clinical human bocavirus can be cultured in differentiated human airway epithelial cells characterization of the gene expression profile of human bocavirus molecular, cellular and clinical aspects of parvovirus b infection nonstructural protein of parvoviruses b and minute virus of mice controls transcription ns protein of parvovirus b interacts directly with dna sequences of the p promoter and with the cellular transcription factors sp /sp minute virus of mice nonstructural protein ns- is necessary and sufficient for trans-activation of the viral p promoter positive and negative regulation of the minute virus of mice p promoter transcriptional activation by the parvoviral nonstructural protein ns- is mediated via a direct interaction with sp efficient trans-activation of the minute virus of mice p promoter requires upstream binding of ns an sp -binding site and tata element are sufficient to support full transactivation by proximally bound ns protein of minute virus of mice characterization of cis-acting and ns protein-responsive elements in the p promoter of parvovirus b key elements of the human bocavirus type (hbov ) promoter and its trans-activation by ns protein this work was supported by the grants from the national nature science foundations of china ( ) and the science and technology foundation of the education department (q ), hubei province, china. the granting agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. statistical analyses were performed with spss, version . . we expressed continuous variables as the median +/− standard deviation. a p value less than . was considered statistically significant. written informed consent was obtained from the patients for the publication of this report and any accompanying images. the authors declare that they have no competing interests.authors' contributions jjl performed the experiments and wrote the first draft of the paper, in collaboration with ymd and ysl. yh and qhy analyzed the data and drafted the manuscript. yl, kyl and yby participated in the design and coordination of the study and revised the manuscript. all authors read and approved the final manuscript. key: cord- -giea b authors: firth, andrew e; atkins, john f title: a case for a cug-initiated coding sequence overlapping torovirus orf a and encoding a novel kda product date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: giea b the genus torovirus (order nidovirales) includes a number of species that infect livestock. these viruses have a linear positive-sense ssrna genome of ~ - kb, encoding a large polyprotein that is expressed from the genomic rna, and several additional proteins expressed from a nested set of '-coterminal subgenomic rnas. in this brief report, we describe the bioinformatic discovery of a new, apparently coding, orf that overlaps the ' end of the polyprotein coding sequence, orf a, in the + reading frame. the new orf has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of orf a. we propose that the new orf utilizes a non-aug initiation codon - namely a conserved cug codon in a strong kozak context - upstream of the orf a aug initiation codon, resulting in a novel amino acid protein, dubbed ' k'. the genus torovirus belongs to the family coronaviridae in the order nidovirales. species include bovine torovirus, equine torovirus and porcine torovirus. as with other members of the order nidovirales, these viruses have a linear positive-sense ssrna genome encoding a large replicase polyprotein that is expressed from the genomic rna (orf a and, via ribosomal frameshifting, an orf a-orf b fusion product), and a number of other proteinsincluding the structural proteins -which are translated from a nested set of '-coterminal sub-genomic rnas ( figure a ) [ ] [ ] [ ] [ ] [ ] [ ] . overlapping genes are common in rna viruses where they serve as a mechanism to optimize the coding potential of compact genomes. however, annotation of overlapping genes can be difficult using conventional genefinding software [ ] . recently we have been using a number of complementary approaches to systematically identify new overlapping genes in virus genomes [ ] [ ] [ ] [ ] [ ] . when we applied these methods to the toroviruses, we found strong evidence for a new coding sequence -overlapping the '-terminal region of orf a ( figure ). here we describe the bioinformatic analyses. relatively little sequence data is available for the relevant '-terminal region of the torovirus genome. in fact there are only two non-identical sequences in genbank (tblastn [ ] [ ] , and [genbank:dq ] -berne virus or equine torovirus [ ] . however these two viruses are reasonably divergent (mean nucleotide identity within orf ã %), thus providing robust statistics for comparative methods of gene prediction. the nc_ and coding potential statistics for torovirus orf a and the overlapping orfx [ ] for details). (b ) depicts the probability that the degree of conservation within a given window could be obtained under a null model of neutral evolution at synonymous sites, while (b ) depicts the absolute amount of conservation as represented by the ratio of the observed number of substitutions within a given window to the number expected under the null model. note that the relatively large sliding window size ( codons) -used here for improved statistical power -is responsible for the broad smoothing of the conservation scores at the ' end of orfx. (b -b ) mlogd sliding-window plots (window size codons; step size codons; see [ ] for details). the null model, in each window, is that the sequence is non-coding, while the alternative model is that the sequence is coding in the given reading frame. positive scores favour the alternative model and, as expected, in the + frame (b ) there is a strong coding signature throughout orf a except where orf a is overlapped by orfx (see text). in the + and + frames (b -b ), scores are generally negative, albeit with significant scatter into positive scores (a reflection of the limited amount of available input sequence data). nonetheless the orfx region is characterized by consecutive positively scoring windows in the + frame (b ). note that, regardless of the sign (either positive or negative), the magnitude of mlogd scores tends to be lower within the overlap region itself (b -b ) due to there being fewer substitutions with which to discrimate the null model from the alternative model in this region of above-average nucleotide conservation. dq orf a amino acid sequences were aligned with clustalw [ ] and back-translated to produce a nucleotide sequence alignment, which was analyzed with a number of techniques. the first piece of evidence for an overlapping coding sequence is the presence of an unusually long open reading frame ( codons; hereafter orfx) at the ' end of orf a but in the + reading frame relative to orf a (figure b, panels - ) . in fact orf a in breda virus has stop codons in the + frame (out of a total of codons), while berne virus has stop codons (out of ). in other words, approximately one in every eight codons in the + reading frame is a stop codon (see, for example, the last three alignment blocks in figure ). thus the probability of obtaining an uninterupted -codon + frame orf simply by chance is vanishingly small (if + frame stop codons within orf a are assumed to be randomly distributed, then the probability is of order p < - ). moreover, there are point nucleotide differences between breda virus and berne virus within orfx, and yet the open reading frame is preserved in both viruses. the absence of stop codons may be linked to local nucleotide biases -indeed the mean nucleotide frequencies within orfx (breda virus) are a %, c %, g % and u % compared with a %, c %, g % and u % in the rest of orf a, so that the orfx region is relatively c-rich and u-poor. however the simplest explanation for these nucleotide biases is simply the presence of an overlapping gene (i.e. orfx) and the constraints imposed by having to code in multiple reading frames. next, the orf a alignment was analysed for conservation at synonymous sites, as described in [ ] (but inspired by ref. [ ] ). the procedure takes into account whether synonymous site codons are -, -, -, -or -fold degenerate and the differing probabilities of transitions and transversions. there was a striking, and highly statistically significant (p < - for the total conservation within orfx), peak in orf a-frame synonymous site conservation at the ' end of the alignment, corresponding precisely to the conserved open reading frame, orfx ( figure b , panels - ). peaks in synonymous sites conservation are generally indicative of functionally important overlapping elements, though such elements may be either coding or non-coding. in fact, high synonymous site conservation at the ' end of long polyprotein-encoding sequences is a feature common to a number of rna viruses and can not, in itself, be taken as evidence of an overlapping coding sequence. however the extent ( codons) and degree ( figure b, panel ) of the conservation here is unusual and, furthermore, the high conservation is not matched in the related coronaviruses. thus an overlapping gene, viz. orfx, provides the most obvious explanation for the high conservation seen here. (an alternative explanation is recombination, as in ref. [ ] . however recombination does not provide an explanation for the other evidence presented in this report.) finally, we analysed the alignment with mlogd -a genefinding program which was designed specifically for identifying overlapping coding sequences, and which includes explicit models for sequence evolution in multiply-coding regions [ , ] ( figure b, panels - ) . in contrast to the synonymous site conservation index above, mlogd, when applied in the sliding window mode, does not depend on the degree of conservation per se (the sequence divergence parameter is fitted independently for each window). with just two input sequences, the mlogd signal proved to be somewhat noisy (e.g. there are a number of positively scoring windows that clearly do not correspond to potential overlapping genes in, for example, the + frame; figure b , panel ). however the signal for orfx was clear -with consecutive positively scoring windows throughout the orfx region in the + frame -indicating, again, that orfx is indeed a coding sequence. moreover, the mlogd score in the + /orfx frame within the orfx region was significantly greater than the score in the + / orf a frame, indicating that the orfx product is subject to stronger functional constraints than the product of the overlapping region of orf a (which indeed has a negative mlogd score towards the '-terminal half of the orfx region). consistently, further inspection showed that, in the region where orfx and orf a overlap, orfx has higher amino acid conservation than orf a ( / identities for orfx, / identities for orf a). in breda virus (nc_ ), the annotated orf a aug initiation codon is at nucleotide coordinates .. and the first orfx-frame aug codon is at coordinates .. . however leaky scanning to this aug codon is unlikely, due to intervening aug codons in the orf a frame ( in nc_ , in dq ; figure ). instead we propose that orfx initiation takes place at a cug codon located upstream of the orf a aug codon, at coordinates .. (figure ). cug is, apparently, the most commonly used non-aug initiation codon in mammalian systems (reviewed in [ ] ), and this particular cug codon is conserved, and has a strong kozak context ('a' at - , 'g' at + ; [ ] ), in both breda and berne viruses. the downstream sequence is predicted to fold into a hairpin structure that is identical between breda and berne viruses -despite a number of base variationsand that is separated from the cug codon by nt (figure ) . such structures -particularly at this spacing -have been shown to greatly enhance initiation at non-aug codons [ ] . moreover, inspection of the sequence alignment upstream of the orf a initiation site shows that the majority ( orfx-frame coding sequence (figure ). this pattern of base variation continues right up to the proposed cug initiation codon. initiation at a site further upstream is precluded by orfx-frame termination codons and, consistently, the sequence further upstream does not maintain the reading frame and base variations no longer favour the rd position ( figure ). initiation at the upstream cug codon would give orfx the nucleotide coordinates .. in nc_ and .. in dq , resulting in a amino acid product with a molecular mass of kda which, for want of a better designation, we tentatively name ' k'. the full predicted amino acid sequences are shown in figure . note that the product has only one methionine residue, making detection with [ s]met difficult. application of blastp [ ] to the amino acid sequences revealed no similar sequences in genbank ( aug ) -as expected for a gene created de novo via out-of-frame 'overprinting' of a preexisting gene [ , ] . similarly, application of inter-proscan [ ] also returned no hits (protein motifs, domains etc). it is expected that a large proportion of ribosomes should scan past the cug codon and initiate at the orf a aug codon -thus allowing synthesis of the replicase polyprotein -though the additional possibility that the cug-initiation efficiency may be temporally regulated as part of the virus lifecycle can not currently be discounted [ , ] . overlapping genes are difficult to identify and are often overlooked. however, it is important to be aware of such genes as early as possible in order to avoid confusion (otherwise functions of the overlapping gene may be wrongly ascribed to the gene they overlap), and also so that the functions of the overlapping gene may be investigated in their own right. we hope that presentation of this bioinformatic analysis will help fullfil these goals. initial verification of orfx product could be by means of immunoblotting with orfx-specific antibodies, bearing in mind, however, that it may be expressed at relatively low levels. toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily amino acid alignment for ' k', the translated orfx figure amino acid alignment for ' k', the translated orfx. note, here the proposed cug initiation codon is assumed to be translated by initiator met-trna -resulting in an n-terminal methionine rather than leucine. . hoet ae, saif lj: bovine torovirus (breda virus) revisited discontinuous and non-discontinuous subgenomic rna transcription in a nidovirus characterization of a torovirus main proteinase the complete sequence of the bovine torovirus genome nidovirus transcription: how to make sense detecting overlapping coding sequences with pairwise alignments detecting overlapping coding sequences in virus genomes an overlapping essential gene in the potyviridae discovery of frameshifting in alphavirus k resolves a -year enigma a conserved predicted pseudoknot in the ns a-encoding sequence of west nile and japanese encephalitis flaviviruses suggests ns ' may derive from ribosomal frameshifting basic local alignment search tool clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice goodfellow ig: bioinformatic and functional analysis of rna secondary structure elements among different genera of human and animal caliciviruses phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events generation of protein isoform diversity by alternative initiation of translation at non-aug codons an analysis of '-noncoding sequences from vertebrate messenger rnas downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes the evolution of genome compression and genomic novelty in rna viruses overlapping genes produce proteins with unusual sequence properties and offer insight into de novo protein creation interproscan -an integration platform for the signature-recognition methods in interpro uorfs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs this work was supported by national institutes of health grant r gm and an award from science foundation ireland, both to jfa. the authors declare that they have no competing interests. aef carried out the bioinformatic analysis and wrote the manuscript. both authors edited and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge key: cord- -iq xqrn authors: obeng, billal musah; bonney, evelyn yayra; asamoah-akuoko, lucy; nii-trebi, nicholas israel; mawuli, gifty; abana, christopher zaab-yen; sagoe, kwamena william coleman title: transmitted drug resistance mutations and subtype diversity amongst hiv- sero-positive voluntary blood donors in accra, ghana date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: iq xqrn background: detection of hiv- transmitted drug resistance (tdr) and subtype diversity (sd) are public health strategies to assess current hiv- regimen and ensure effective therapeutic outcomes of antiretroviral therapy (art) among hiv- patients. globally, limited data exist on tdr and sd among blood donors. in this study, drug resistance mutations (drms) and sd amongst hiv- sero-positive blood donors in accra, ghana were characterized. methods: purposive sampling method was used to collect hiv sero-positive blood samples from the southern area blood center and confirmed by inno-lia as hiv- and/or hiv- . viral rna was only extracted from plasma samples confirmed as hiv- positive. complementary dna (cdna) was synthesized using the rna as a template and subsequently amplified by nested pcr with specific primers. the expected products were verified, purified and sequenced. neighbour-joining tree with the kimura’s -parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis version . (mega . ). results: out of the plasma samples, ( %) were confirmed as hiv- sero-positive by inno-lia hivi/ii score kit with no hiv- and dual hiv- / infections. the remaining samples, ( %) were confirmed as hiv sero-negative. of the confirmed positive samples, ( ) % and ( ) % were successfully amplified in the rt and pr genes respectively. nucleotide sequencing of amplified samples revealed the presence of major drug resistance mutations in two ( ) samples; e a in one sample and another with k r. hiv- subtypes including subtypes a, b, crf _ag and crf _cpx were found. conclusion: this study found major drug resistance mutations, e a and k r in the rt gene that confer high level resistance to most nnrtis and nrti respectively. crf _ag was most predominant, the recorded percentage of subtype b and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. however, a more prospective and detailed analysis is needed to establish this phenomenon. the data obtained would inform the selection of drugs for art initiation to maximize therapeutic options in drug-naïve hiv- patients in ghana. the hiv global burden is estimated at . million infected persons with approximately million living in sub-saharan africa [ ] . hiv- infection was first detected in ghana in and has since been responsible for numerous deaths of both children and adults [ ] . currently, the prevalence of hiv- in ghana is estimated to be . % in [ ] . a major therapeutic intervention to the hiv pandemic has been the introduction and access to antiretroviral therapy (art) [ ] . this has reduced hiv-related morbidity and mortality rates and increased life expectancy of infected individuals [ ] . however, therapeutic success of these arts is reduced as a result of emergence of hiv- drms, viral sd and spontaneously generated polymorphisms due to immune pressure in patients receiving arts [ ] . the absence of genotypic drug resistance monitoring may lead to patients harboring viruses with drm which could be transmitted to new hosts, a phenomenon described as transmitted drug resistance (tdr) [ ] . tdr is an important public health concern due to increased risk of virologic failure when art is initiated [ ] . systematic studies have suggested higher rates of tdr ( - %) in europe, america, and australia, as compared to countries where scale-up of art is ongoing [ ] [ ] [ ] . in these studies, the prevalence of tdr in nucleoside reverse transcriptase inhibitors (nrti), nonnucleotide reverse transcriptase inhibitors (nnrti) and protease inhibitors (p ), were . , . and % respectively. comprehensive care for people living with hiv/aids (plhiv) in ghana started in with first-line drugs including azidothymidine (azt), stavudine (d t), lamivudine ( tc), nevirapine (nvp) and efavirenz (efv). over the past decade, these drugs have expanded to include emtricitabine (ftc), tenofovir (tdf), and abacavir (abc). since its inception, art has been scaled up to about %of hiv patients [ ] . limited data has shown that drm and tdr in naïve plhiv in ghana are low [ ] [ ] [ ] [ ] . a threshold survey among pregnant women in the administrative region where art was first introduced in ghana observed a tdr rate of < % [ ] . voluntary blood donors (vbd) provide a population base which is young and are more likely to have been recently exposed to hiv [ ] . in order to understand the transmission dynamics among newly exposed people living with hiv (plhiv), tdr in a cross-section of vbd needs to be assessed. a cross-sectional study using purposive sampling method was conducted at the southern area blood center (sabc), ghana from august to february . the center is a satellite facility of the national blood service, ghana (nbs). it is mandated to collect, screen, and distribute safe blood to various hospitals and clinics in the southern part of ghana. the nbs recruits vbd using a questionnaire to first assess their behavioral risk factors and health status to ascertain their suitability to donate blood. upon successful recruitment and blood donation, the blood is screened for hbsag, hcv antibodies, hiv and antibodies for treponema pallidum as procedures for safe blood. the test algorithm for hiv- at the blood bank is by the detection of p antigen using the hiv (ag/ab) thgen (fortress diagnostics limited, antrim, u.k). currently, a more sensitive test tool such as the pcr and/or inno-lia is not employed. ethical approval was obtained from the ethics and protocol review committee of the college of health sciences, university of ghana. approval to select hiv positive samples was also obtained from the nbs. a total of eighty-one ( ) voluntarily donated blood samples that were rejected as been hiv sero-positive using the hiv (ag/ab) thgen (fortress diagnostics limited, antrim, u.k) were used. a data extraction sheet was used in obtaining information on age and gender from the donors' records upon approval from the sabc. study numbers were assigned to anonymize the blood samples. plasma was obtained from the sabc and were transported in cold boxes with ice packs to the virology department of nmimr and stored at − °c until further processing. a confirmatory test (inno-lia™ hiv-i/ii score, fujirebio, gent, belgium) was done on all plasma samples following manufacturer's protocol. viral rna was extracted using the qiaamp® viral rna mini kit (qiagen, hilden, germany) following manufacturer's protocol. a two-step reverse transcription method was used to generate complementary dna (cdna) of hiv- from extracted rna using transcriptor high fidelity cdna synthesis kit (roche diagnostics, mannheim, germany). an initial reaction mix of . μl random hexamer primer, . μl nuclease-free water and . μl of extracted rna were incubated at °c for min and immediately placed on ice. a second reaction mix made up of . μl of x high fidelity reverse transcriptase ( x hfrt) buffer, . μl protector rnaase inhibitor, . μl deoxynucleotide triphosphates (dntps), . μl dithiothreitol (dtt) and . μl transcriptor hfrt enzyme was prepared. an aliquot of . μl of the second mix was added to the first reaction, thoroughly mixed and incubated at °c for min followed by °c for min. nested pcr was done to separately amplify the protease (pr) and reverse transcriptase (rt) genes from the cdna synthesized using the expand high fidelity plus pcr kit (roche diagnostics, mannheim, germany) with specific primers and cycling conditions previously published [ ] . in the first round, . μl of × buffer with mgcl , . μl of dntps, . μl each of forward and reverse primers, . μl of expand high fidelity polymerase and . μl of nuclease free water were added to . μ l of cdna. in the second round of the pcr, . μl of x buffer with mgcl , . μl dntps, . μl of each of forward and reverse primers, . μl expand high fidelity polymerase and . μl nuclease-free water were added to . μl of round product. a fragment of base pairs (bp) and bp for the pr and rt genes respectively, were generated and confirmed by agarose gel electrophoresis. purification of nested pcr products was done using qiaquick pcr purification kit (qiagen, hilden, germany) following manufacturer's protocol. purified amplicons were eluted in μl of elution buffer for cycle sequencing. the bigdye terminator v . cycle sequencing kit (applied biosystems, ma, u.s.a) was used to separately sequence the pr and rt genes of hiv- using primers and cycling conditions previously published [ ] . a total reaction volume of μl comprising of μl each of a primer, bigdye terminator, bigdye terminator buffer, nuclease free water and purified pcr product was used. sequenced products were purified using the agencourt® cleanseq® dye-terminator removal system (agencourt bioscience corporation, u.s.a) following manufacturer's protocol. the purified product was loaded onto the abi xl genetic analyzer (applied biosystems, ma, u.s.a) to generate sequence data for hiv- drm and hiv subtype analyses. nucleotide sequences for each sample were assembled to form a contig using seqmanpro (dnastar incorporation, u.s.a). consensus sequence obtained was aligned with an hiv reference sequence (b-hxb -prt_ - ) in bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). sequences were submitted to the stanford university hiv drug resistance database (https:// hivdb.stanford.edu/hivdb/by-sequences/) to assign subtypes detect hiv drug resistance mutations and. the subtypes were confirmed with the los alamos national laboratory hiv database (http://www.hiv.lanl.gov). neighbor-joining tree with the kimura's -parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis version . (mega ). statistical analysis was done using spss version (armonk, usa) to describe patients' demographics using frequencies and percentages. a total of eighty-one ( ) hiv- sero-positive blood samples were selected from the blood bank for this study. the hiv status, age and gender characteristics of participants is summarized in table . majority ( %) were between the ages of to years with to year group being the least recorded ( %). majority ( %) were males whilst % were females. age and gender information for ( %) of the study samples were not available in the sabc donor records. sixty ( %) of the samples were confirmed as hiv- positive, twenty-one samples ( %) were found to be negative for hiv- , hiv- or dual hiv- / infection. of the samples confirmed reactive for hiv- , ( %) and ( %) were successfully amplified for the pr and rt genes respectively. of these, / ( %) and / ( %) were successfully sequenced for the pr and rt genes respectively. eight samples had sequences for both the pr and rt genes. one ( ) sample had minor drm in the pr gene whilst minor and major drms were found in the rt gene of three ( ) samples as shown in table . out of the pr sequences, ( %) was subtype a, ( %) were subtype b and ( %) were crf _ag. of the rt sequences, ( %) were subtype a, ( %) were subtype b, ( %) were crf _ag and ( %) was crf _cpx as indicated in fig. . no major pr-related drm was observed in the samples whilst major rt-related drms in two ( ) samples were found; e a in one sample and another with k r. drug resistance implications of the mutations found are shown in table . phylogenetic analysis using the neighbour-joining tree with the kimura's -parameter distances was generated with the rt sequences using molecular evolutionary genetic analysis tool version . (mega ). study samples were labelled with coloured squares (red, green, blue and black squares indicating different subtypes) and reference sequences labelled with accession numbers as shown in fig. . this study examined occurrence of drug-resistance mutations in apparently healthy blood donors who tested positive for hiv- .the proportion of males to females in this study does not concord with hiv- trends. the same could be said for the distribution of hiv infection across the age groups. although, the number of females infected with hiv nationwide is more than males [ ] , voluntary blood donors are usually males. females are usually disqualified due to relatively lower hemoglobin (hb) levels. this is partly because of monthly blood loss during menstruation and the toll of pregnancy. moreover, it is physically active men usually between the ages of - years who volunteer to donate blood. thus, these reasons could have accounted for the pattern seen in this study. the predominance of crf _ag and absence of hiv- and dual hiv- / infections agrees with studies reporting hiv- infection as the predominant hiv type in ghana and crf _ag as the main subtype [ , ] . the twenty ( ) samples that were later confirmed to be hiv negative indicate the need for diagnostic methods with higher sensitivity at blood banks to minimize the probability of transfusing infected blood [ ] . hiv- tdr occurs when recently infected individuals who are not exposed to antiretroviral drugs harbor drug resistant viruses. in this study, hiv- tdr is defined as the presence of at least one major hiv- drm in a study participant. two ( ) major drms were found in two ( ) samples sequenced in the rt gene (e a and k r) and none in the pr gene. other mutations were also found, which do not confer drug resistance by themselves but only when they occur in combination with other mutations. the accessory and minor mutations found were f l and l f in rt gene and pr gene respectively. the e a mutation is a polymorphic mutation that occurs in an appreciable number of drug-naïve patients such as the population studied and confers resistance to etravirine (etr) and rilpivirine (rpv), which are nnrt is [ , ] . in a similar study, e a was found in artnaïve pregnant women attending antenatal care in a teaching hospital in accra, ghana [ ] . this mutation, has been shown to reduce susceptibility to etr and rpv by -folds. the k r mutation is found to reduce viral susceptibility to most nrtis by approximately fold and rather increase susceptibility to azt and hence reduced viral replication with zidovudine-containing therapy [ ] . the findings of this study are consistent with other studies previously conducted among drug naïve infected individuals, in which pi-associated drm were rarely found [ , ] . generally, low level drms against pis in the ghanaian population could be attributed to the high genetic barrier of protease inhibitors and that the virus would have to mutate several times to develop resistance to such drugs. additionally, sparing use of protease inhibitors reserved for use mostly in second-line regimen while majority of those on treatment are on reverse transcriptase inhibitors may have accounted for this phenomenon. viral sequence subtyping showed crf _ag as most predominant subtype in the study population. other subtypes found were b, a and crf _cpx. this result is similar to some studies conducted in west africa [ , ] and in ghana [ , ] . the predominance of crf _ag has been associated with high viral infectivity and productivity, possible replicative fitness and high viral loads which favour viral transmission [ , ] . subtype b was the next predominant subtype and was relatively frequent compared to previous studies in ghana [ ] . subtype b is most predominant in the americas [ , ] and western europe [ , ] . the occurrence of hiv- subtypes in different geographical areas has been linked to socio-epidemiologic factors such as mobility and migration [ , ] . these factors, however, cannot be confirmed for this fig. percentage occurrence of hiv- subtypes in sequenced pr and rt genes. figure shows hiv- subtype patterns in the pr and rt genes. pr and rt sequences were submitted online to the stanford university hiv drug resistance database (hivdb). one ( %) was subtype a, ( %) were subtype b and ( %) were crf _ag. of the rt sequences, ( %) were subtype a, ( %) were subtype b, ( %) were crf _ag and ( %) was crf _cpx. key: pr protease, rt reverse transcriptase, crf circulating recombinant form the study observed low amplification rate of samples. this could be due to low viral loads in some samples. however, it is not entirely the reason for low amplification rate as samples with low viral loads were successfully amplified whilst others with higher loads were not amplified. previous research shows that samples from patients with persistently low viral load could be genotyped by a nested pcr method [ , ] . the inability to obtain peripheral blood mononuclear cells (pbmc) to amplify alongside the plasma could also account for a lower amplification rate. amplification success with proviral dna from pbmc was found to be relatively higher than viral rna from plasma [ , ] . genotyping from plasma rna and proviral dna concurrently could have increased amplification success since some plasma samples may be amplified and not their pbmc samples and vice versa. despite these limitations, the study obtained data that is important for hiv management in ghana. this study found major drug resistance mutations, e a and k r that respectively confer high level resistance to nnrtis and nrtis. although, crf _ag was most predominant, the recorded percentage of subtype b and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. a more prospective and detailed analysis is needed to establish this phenomenon. the data obtained is useful for the selection of drugs for art initiation to maximize therapeutic outcomes in drug-naïve hiv- patients in ghana. continuous surveillance for drug resistance mutations and subtype diversity in population groups is therefore imperative for effective art outcomes. we thank the national blood service, ghana for the samples used in this study. technical support by the hiv genotyping team of noguchi memorial institute for medical research is acknowledged. the hiv- rt and pr sequences have been deposited at genbank under accession numbers mn -mn . the data sets are available with the corresponding author on request. the data sets used and analysed during this study are available with the corresponding author on request. ethics approval and consent to participate the protocol was approved by the ethics and protocol review committee of the college of health sciences, university of ghana (chs-et/m. -p . / - ). approval to select hiv positive samples was also obtained from the nbs (nbsgrd/ / ). the nbs has given the consent to publish the results without personal identification. background, projections, impacts, interventions and policy hiv- drug resistance-associated mutations among hiv- infected drug-naïve antenatal clinic attendees in rural kenya declining morbidity and mortality among patients with human immunodeficiency virus infection: hiv outpatient study investigators the prevalence of antiretroviraldrug resistance in the united states transmitted hiv- drug resistance in africa. lusaka: th interest workshop effect of transmitted drug resistance on virological and immunological response to initial combination antiretroviral therapy for hiv (eurocoord-chain joint project): a european multicohort study report on the global epidemic the epidemiology of antiretroviral drug resistance among drug-naive hiv- -infected persons in us cities prevalence of drug-resistant hiv- variants in untreated individuals in europe: implications for clinical management, spread programme variability of the human immunodeficiency virus type polymerase gene from treatment naive patients in accra, ghana genotypic diversity and mutation profile of hiv- strains in antiretroviral treatment (art) -naïve ghanaian patients and implications for antiretroviral treatment (art) low level of transmitted hiv drug resistance at two hiv care centres in ghana: a threshold survey hiv- drug-resistance surveillance among treatment-experienced and treatment-naıve patients after the implementation of antiretroviral therapy in ghana update: active involvement of young people is key to ending the aids epidemic by improved conditions for extraction and amplificationof human immunodeficiency virus type rna from plasma samples with low viral load performance and quality assurance of genotypic drug-resistant testing for human immunodeficiency virus type in japan epidemiologic dominance of hiv- subtype crf _ag in ghana: preliminary virological evidence of increasing association with new infections higher viral load may explain the dominance of crf _ag in the molecular epidemiology of hiv in ghana impact of hiv- reverse transcriptase e mutations on rilpivirine drug susceptibility effect of mutations at position e in hiv- reverse transcriptase on phenotypic susceptibility and virologic response to etravirine occurrence of transmitted hiv- drug resistance among drug-naïve pregnant women in selected hiv care centers in ghana intensification of a failing regimen with zidovudine may cause sustained virologic suppression in the presence of re-sensitizing mutations including k r prevalence and determinants of transmitted antiretroviral drug resistance in hiv- infection predominance of crf -ag and crf -cpx in niger, west africa the predominance of human immunodeficiency virus type (hiv- ) circulating recombinant form (crf _ag) in west central africa may be related to its replicative fitness genetic analysis of hiv- isolates from brazil reveals the presence of two distinct genotypes genetic diversity of the envelope glycoprotein from human immunodeficiency virus type isolates of african origin hiv- subtypes in luxembourg surveillance of hiv- subtypes among heterosexuals in england and wales tracking a century of global expansion and evolution of hiv to drive understanding and to combat disease the origin and diversity of the hiv- pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations no competing interest exist. key: cord- - s q c authors: filoni, claudia; helfer-hungerbuehler, a. katrin; catão-dias, josé luiz; marques, mara cristina; torres, luciana neves; reinacher, manfred; hofmann-lehmann, regina title: putative progressive and abortive feline leukemia virus infection outcomes in captive jaguarundis (puma yagouaroundi) date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: s q c background: feline leukemia virus (felv) is an exogenous gammaretrovirus of domestic cats (felis catus) and some wild felids. the outcomes of felv infection in domestic cats vary according to host susceptibility, virus strain, and infectious challenge dose. jaguarundis (puma yagouaroundi) are small wild felids from south and central america. we previously reported on felv infections in jaguarundis. we hypothesized here that the outcomes of felv infection in p. yagouaroundi mimic those observed in domestic cats. the aim of this study was to investigate the population of jaguarundis at fundação parque zoológico de são paulo for natural felv infection and resulting outcomes. methods: we investigated the jaguarundis using serological and molecular methods and monitored them for felv-related diseases for years. we retrieved relevant biological and clinical information for the entire population of jaguarundis held at zoo. post-mortem findings from necropsies were recorded and histopathological and immunohistopathological analyses were performed. sequencing and phylogenetic analyses were performed for felv-positive samples. for sample prevalence, % confidence intervals (ci) were calculated. fisher’s exact test was used to compare frequencies between infected and uninfected animals. p-values < . were considered significant. results: in total, we detected evidence of felv exposure in four out of animals ( %; % ci – %). no endogenous felv (enfelv) sequences were detected. an intestinal b-cell lymphoma in one jaguarundi was not associated with felv. two jaguarundis presented felv test results consistent with an abortive felv infection with seroconversion, and two other jaguarundis had results consistent with a progressive infection and potentially felv-associated clinical disorders and post-mortem changes. phylogenetic analysis of env revealed the presence of felv-a, a common origin of the virus in both animals ( % identity) and the closest similarity to felv-faids and felv- ( . % identity), originally isolated from cats in the usa. conclusions: we found evidence of progressive and abortive felv infection outcomes in jaguarundis, and domestic cats were probably the source of infection in these jaguarundis. jaguarundis (puma yagouaroundi) are small diurnal felids that have several unpatterned color morphsbrownish-black, gray and reddish-yellow furand are protected across most of their range. the species occurs at low densities and has a decreasing population in the wild, despite being widely distributed throughout south and central america and occupying a broad range of habitats [ ] . mainly due to displacement from nature and, to a lesser extent, to captive breeding, jaguarundis are commonly found in zoos and similar captive settings in brazil [ ] . the feline leukemia virus (felv) is an exogenous, oncogenic, immunosuppressive gammaretrovirus that can establish persistent infections in domestic cats (felis catus) [ ] . the prevalence of felv ranges from % to % in healthy cats almost everywhere in the world and is usually higher when sick cats are included [ ] . felv is naturally transmitted by oronasal exposure to viruscontaining secretions, mainly saliva, but also in feces and urine [ ] [ ] [ ] . the effects of felv are cytoproliferative diseases including lymphomas and myeloproliferative disorders; degenerative illnesses, such as anemia and leukopenia; and immunosuppressive diseases associated with opportunistic infections [ , ] . felv infections tend to be rare or absent in many nondomestic felid species [ ] [ ] [ ] [ ] [ ] [ ] , except for the european wildcat (felis silvestris silvestris), a species very closely related to the domestic cat, in which felv appears to be endemic [ ] [ ] [ ] . however, documentation of felv is becoming more common in wild felid species less closely related to the genus felis, which highlights the omnipresent threat that felv represents to the conservation of wild felids worldwide. felv has been shown to represent a major threat to the survival of critically endangered populations of iberian lynxes (lynx pardinus) in europe [ , ] and to florida panthers (puma concolor coryi) in north america [ , ] . in brazil, antibodies against felv have been detected in two freeranging pumas (puma concolor) and two jaguarundis; felv dna was detected in the following captive felids: an ocelot (leopardus pardalis), an oncilla (leopardus tigrinus) and two jaguarundis [ ] [ ] [ ] . for domestic cats, the detection of the structural viral protein felv p in serum or plasma is used as a marker of infection and, in most cases, as a parameter for viremia. the outcomes of felv infection vary according to infectious challenge dose, route of challenge, and possibly host susceptibility and virus strain. the classification of felv outcomes in the domestic cat has been refined using sensitive molecular assays that detect and quantify proviral felv dna and viral felv rna, in addition to traditional serological and virological methods [ ] [ ] [ ] [ ] [ ] [ ] . in this regard, the main host response categories of felv infection in domestic cats have been redefined as abortive, regressive, and progressive infection. in short, those cats that abort infection do not show any evidence of virus infection, except for seroconversion. cats with regressive infection overcome viremia after an undetectable or transient initial phase by means of efficient cellular and humoral immune responses. these cats seroconvert, permanently harbor low to moderate felv proviral loads integrated in mononuclear cells, i.e., lymphocytes, and may or may not clear their plasma viral rna loads [ , , ] . cats with progressive infection are constantly viremic, have low or no antibodies to felv, show elevated felv proviral and viral loads in peripheral blood cells and plasma and may develop felv-associated disease [ , , , ] . in previous surveillance work, we detected felv infection in two captive-born jaguarundis (# and # ) as well as previous exposure to felv in two other captive-born jaguarundis (# and # ) among a population of jaguarundis held at fundação parque zoológico de são paulo (fpzsp), brazil [ ] . jaguarundis # and # tested positive for felv p antigen by sandwich elisa. felv proviral dna was detected in blood from both animals by quantitative real-time polymerase chain reaction (qpcr). the sampling for this previous study occurred between and (table ) . by analogy, we hypothesized here that felv infection in p. yagouaroundi mimics the main outcomes observed for the domestic cat. thus, the aim of this study was to perform additional serological and molecular tests and monitor the population of jaguarundis at fpzsp for felv infection and development of felv-related diseases for years ( ) ( ) ( ) ( ) ( ) . we retrieved relevant biological and clinical information for the entire population of jaguarundis held at fpzsp, from birth or admission of the animals into the zoo until . most animals were mature adults (n = ), five were immature and two were geriatric; kittens were absent. three jaguarundis were wild born, while were captive born at fpzsp. six females (two of wild origin) and three zoo-born males were reproductively active. eleven animals from the population were full siblings, and were half siblings. the females (n = ) averaged . ± . kg in body weight, and the males (n = ) averaged . ± . kg in body weight. detailed biological data such as age, sex, weight, origin (captive or wild born) and the parental history for the jaguarundis are presented in table . for hematological analysis, several laboratories were used over time, and registries of results from the animals were not completed and standardized. all jaguarundis notes: felv-infected (# , # ) and felv seropositive (# , # ) jaguarundis are identified in bold. a for the wild-born jaguarundis (# , # and # ), the dates refer to entrance in the zoo as their dates of birth are unknown. b immature adults (n = ) were defined as jaguarundis in the age range of months ≤ age < months for males and months ≤ age < months for females; mature adults (n = ) were defined as those in the age range of months ≤ age < years ( months) for males and months ≤ age < years for females; geriatric jaguarundis (n = ) were those aged ≥ years for both sexes. c for two captive-born jaguarundis (# , # ), the identities of the father and the mother, respectively, were not available were vaccinated regularly against feline herpesvirus (fhv- ), feline calicivirus (fcv), feline parvovirus (fpv) and rabies lyssavirus. we monitored the potential development of felv-related diseases through clinical data provided by the veterinarian staff at the zoo and registered at the zoo archives. serological and molecular felv tests were performed, in addition to those previously published [ ] , using samples that had been previously obtained or additional samples collected throughout the observation period. felv tests previously performed included p felv antigen detection by a sandwich elisa and the commercial immunoassay snap™ combo felv antigen/fiv antibody test kit (idexx laboratories inc., westbrook, me, usa), detection of antibodies against the recombinant non-glycosylated form of the felv gp surface glycoprotein (p ) by indirect elisa, detection of antibodies against felv p , p , p , p , and gp antigens by western blot and detection of felv proviral dna by real-time pcr. we investigated the presence of antibodies to felv transmembrane protein p e in serum samples from six jaguarundis (# , # , # , # , # and # ) using the previously described assay [ , ] . six jaguarundis (# , # , # , # , # and # ) died between and and underwent necropsy at the zoo; for these jaguarundis, relevant clinical and post-mortem findings from necropsies were recorded, and several tissues were collected for analyses. fragments of tissues from these animals were fixed in % formalin, embedded in paraffin wax and sectioned. the sections were stained with hematoxylin-eosin (he) on glass slides for histopathological analyses. tissue specimens from animals # and # (bone marrow, mesenteric lymph node and spleen) were also stored at ≤ − °c. a saliva specimen from animal # was collected at first sampling using a sterile cotton swab immersed in virus transport media, which consisted of phosphate-buffered saline (pbs) balanced salt solution supplemented with . % bovine albumin (bsa), antimicrobial agents ( u/ml penicillin g, u/ml streptomycin, μg/ml fungizone and μg/ml gentamycin) and was kept at ≤ − °c. no additional material could be obtained from animal # . to clarify the histogenesis of an intra-abdominal mass of tissue that developed in jaguarundi # , we performed immunohistologic assays by the streptavidin-biotinperoxidase complex technique with a commercial immunoperoxidase kit (lsab kit, dako, glostrup, denmark) according to the manufacturer's protocol. we used silanized microscope slides to produce histologic slides, which were processed by usual deparaffination techniques in xylol and hydrated in alcohols (absolute, %, %, and distilled water). a microwave antigen retrieval technique using edta buffer, ph . , was applied [ ] . the sections were incubated overnight at °c with primary antibodies against the t-cell marker cd (rabbit polyclonal antibodies a ; dako; diluted in ), the b-cell marker cd (mouse monoclonal antibodies; clone hm ; m ; dako; diluted in ), and vimentin (mouse monoclonal antibodies; m ; dako; diluted in ) as well as antibodies against proliferating cell nuclear antigen antibodies (pcna) (mouse monoclonal antibodies; m ; dako; diluted in ). for all reagents, negative controls were generated by substituting the primary antibody with a class-matched immunoglobulin. these procedures were conducted using the logistics of the department of pathology, school of veterinary medicine and animal sciences, university of são paulo (usp), são paulo, sp, brazil. we investigated the presence of felv antigens in several paraffin-embedded tissues from the deceased jaguarundis # , # , # and # . this investigation was carried out using the methods and logistics of the institute of veterinary pathology, university of giessen, giessen, germany [ ] . total nucleic acid (tna) was extracted from μl of edta-anticoagulated whole blood or buffy coat or from μl of edta-anticoagulated plasma using the magna pure lc total nucleic acid isolation kit (roche diagnostics, rotkreuz, switzerland). tna was eluted into μl of elution buffer and stored at − °c until pcr testing was performed. rna was extracted from the saliva, plasma samples collected from jaguarundi # and from serum samples from jaguarundis # and # using the qiamp® viral rna mini kit (qiagen). gdna and rna were extracted from tissues (bone marrow, mesenteric lymph node and spleen) from animals # and # upon necropsy, as previously described [ , ] . during all extractions, negative controls consisting of μl of pbs were concurrently prepared with each batch of samples to monitor for cross-contamination. felv viral rna loads and proviral loads were investigated using real-time rt-pcr and pcr and primers targeting the u region of exogenous felv, as previously described [ ] . for tissue samples, the total copy numbers detected per reaction were normalized to the glyceraldehyde -phosphate dehydrogenase (gapdh) gene as described [ ] . felv subgroups a, b and c were investigated in the bone marrow, mesenteric lymph node, and spleen of the jaguarundi # and in the serum and plasma of jaguarundi # by conventional pcr using the felv-a specific primers rb and rb ; the felv-b specific primers rb and rb and the felv-c specific primers rb and rb as described [ , ] . serum from jaguarundi # was tested for the presence of antibodies against feline immunodeficiency virus (fiv) using a commercial immunoassay snap™ combo felv antigen/fiv antibody test kit (idexx laboratories) and by western blotting, as described [ ] . fiv real-time and conventional rt-pcr were performed from whole blood from jaguarundi # , as previously described [ , ] . endogenous felv-like sequences (enfelv) pcr tna samples from the whole blood or buffy coat of all jaguarundis were tested for the presence of endogenous felv (enfelv) by the three qpcr assays enfelv-u - , enfelv-u - , and enfelv-env, as previously described [ ] . felv env sequences were analyzed for the felv-positive jaguarundis # and # from tna of previously collected buffy coats. for comparison, a second sequence obtained from bone marrow of animal # was analyzed . years later, at the time of euthanasia. for the analysis of the full-length felv env sequences, genomic dna from these samples was amplified using pcr with the primers f ( ′ acatatcgtcctcctgaccac ′) and r ( ′ gaaggtcgaaccctggtcaact ′) [ ] , yielding an approximately ´ bp product. to ensure high-fidelity amplification, the pcr was performed using phusion polymerase and hf buffer (finnzyme, ipswich, uk). pcr products were sequenced by microsynth (balgach, switzerland) after purification using the genelute pcr clean-up kit (sigma, fluka gmbh, buchs, switzerland). the jaguarundi felv-a env sequence was submitted to genbank [kr ]. phylogenetic analyses were conducted using mega version [ ] . the felv env sequences were aligned using clustal w [ ] . bootstrap support ( replicates) was calculated by the neighbor-joining (nj) [ ] and maximum parsimony (mp) [ ] methods, and results > % were considered significant [ ] . the mp tree was obtained using the subtree-pruning-regrafting (spr) algorithm [ ] with search level in which the initial trees were obtained by the random addition of sequences ( replicates). all positions containing gaps and missing data were eliminated from the dataset (complete deletion option). for sample prevalence, % confidence intervals (ci) were calculated. fisher's exact test was used to compare frequencies between infected and uninfected animals. pvalues < . were considered significant. the population of jaguarundis kept at fpzsp from to consisted of captive jaguarundis. the animals presented good general condition at sampling. twenty animals presented discrete to moderate oral disorders, including one or more of the following conditions: gingivitis, presence of tartar, periodontitis, tooth loss and/or fractured teeth. no oral disorders were registered in three immature adults, # , # and # . at least once during the experiment, animals presented episodes of diarrhea, jaguarundis demonstrated transitory weight loss and three suffered from dermatological disorders, such as alopecia; for all these clinical conditions, the underlying causes of the problems were unknown. despite regular deworming, intestinal parasites were detected in animals during the study period. in hematological analysis, most animals (n = ) showed some degree of leukopenia (marked: n = ; mild: n = ) and leukocytosis (marked: n = ; mild: n = ). jaguarundi # , which was felv positive, and jaguarundi # , which was felv seropositive, showed mild transient leukocytosis. several (n = ) animals showed mildly increased packed cell volume (pcv) and two showed markedly increased pcv in at least one sampling. jaguarundis # and # showed mildly increased pcv, and jaguarundi # , which was felv seropositive, showed a markedly increased pcv. the increased pcv could be due to dehydration as the animals had been fasted before immobilization. six jaguarundis (# , # , # , # , # and # ) in the investigated population died from to . jaguarundi # was euthanized . years after being diagnosed with felv infection [ ] . this jaguarundi was euthanized because the zoo could not keep it segregated under good ethological conditions, although the animal was in good clinical condition. jaguarundi # developed a large intra-abdominal mass of tissue about year after testing negative for felv. after clinical and ultrasonographic examinations, the animal underwent laparotomy for excision of the mass and died days after surgery due to clinical deterioration. animals # , # , # and # died naturally during the period of study. for the six deceased animals, clinical conditions and main post-mortem findings are presented in table . antibodies against p e antigen were not detected to a level that is considered positive for privately owned domestic cats and the results from all the jaguarundis tested (# , # , # , # , # and # ) were negative (table ) . the excised mass of tissue from jaguarundi # weighed approximately . kg and affected all layers of the wall of the jejunum. in addition, several small foci were present across the mesentery. there was multifocal necrosis as well as intense reactional fibrosis and proliferation of round cells arranged in dense blocks limited by delicate conjunctive septa. the cells were neoplastic and malignant and presented anisokaryosis, anisocytosis, and a high mitotic index. the tumor was found to be an intestinal b-cell lymphoma, as the cells stained positive for a b-cell marker (cd ) but not a t-cell marker (cd ) by immunohistology. no felv rna or provirus was detected in the tissues collected upon necropsy, including tumoral tissues, bone marrow, mesenteric lymph nodes and spleen, and no felv antigens were detected in paraffin-embedded tissues by immunohistological analysis (table ) . because b-cell lymphomas can also be associated with fiv infection, jaguarundi # was tested for fiv by serology and pcr. the animal tested negative for antibodies to fiv in the commercial assay. in the fiv western blot, one band against p was detectable. felv rna and proviral dna were detected in two out of the animals, namely, jaguarundis # and # . from jaguarundi # , a saliva sample was available; it was felv rna positive, indicating shedding of felv at the time of sampling. felv was also demonstrated in the plasma sample collected at a second sampling from jaguarundi # upon necropsy . years later, indicating persistent antigenemia in this animal. for jaguarundis # and # , respectively, the absolute copy numbers of proviral dna were . × and . × copies of dna/ml of buffy coat; the absolute copy numbers of viral rna were . × and . × copies of rna/ml of serum. the results from all felv tests are presented in tables and . furthermore, felv could be demonstrated in the tissue samples collected at necropsy from jaguarundi # , and felv antigens were confirmed by immunohistological analysis of paraffin-embedded tissue samples from deceased jaguarundis # and # . for both jaguarundis, # and # , felv-a was the only felv subtype identified; no felv-b or felv-c was detected. no enfelv was detected in the blood or buffy coat samples from any of the jaguarundis. (fig. ) . when the env surface unit from the felv detected in jaguarundis was compared with those found in iberian lynxes [eu to eu ], a sequence identity of approximately % was obtained (data not shown). we detected evidence of felv exposure in four (# , # , # and # ) out of jaguarundis in the fpzsp ( %; % ci - %). the remaining jaguarundis were negative for felv in all serological and molecular tests performed. the population of jaguarundis presented clinical disorders that were common at fpzsp, as in any other captive setting in brazil [ , ] , and could be associated with a multitude of causes. although the felv-positive jaguarundis also presented some conditions that could be related to felv [ ] , our data were insufficient to prove a causal association. two captive-born male jaguarundis, the geriatric # and the mature adult # , presented serological and molecular felv test results similar to the progressive felv infection outcome in domestic cats [ ] . jaguarundis # and # were both antigenemic. antigenemia is usually used as a measure for viremia in domestic cats and is consistently found in domestic cats with a progressive felv infection outcome [ , ] . additionally, blood, buffy coat, serum and tissue samples from both jaguarundis were positive for felv proviral dna and viral rna with high proviral and viral loads, respectively (table ). while the presence of proviral dna attests to the integration of provirus into blood cells, the detection of viral rna usually indicates replicating virus. once the infection outcome has been established, high proviral and felv rna loads are characteristically found in cats with progressive infection, while low loads are detected in cats with regressive infection that are provirus positive but not viremic at the time [ , , ] . felv proviral and viral rna loads were measured in the two felv-positive jaguarundis # and # . comparison of the felv tissue loads of jaguarundi # revealed loads similar to those reported earlier for domestic cats with progressive infection using identical methods. viral rna loads in serum from the two felv-positive jaguarundis were comparable to those described earlier in persistently antigenemic cats [ ] . the viral rna load in the clinically healthy jaguarundi # was slightly lower than that in jaguarundi # , which showed weight loss, vomiting, anorexia and diarrhea before death. this is also similar to previous findings from a study in cats, comparing viremic healthy and viremic ill cats [ ] . moreover, consistent with findings in domestic cats with a progressive felv infection, no antibodies to felv antigens were detected in jaguarundis # and # . felv rna and felv provirus were also detected in the saliva of jaguarundi # . this finding is indicative of virus shedding; thus, jaguarundi # was a potential source of felv infection to other felids. jaguarundi # was euthanized in good clinical condition as a biosafety measure. however, upon necropsy, histopathologic evaluation revealed splenic lymphoid depletion and muscular atrophy of the hind limbs; these findings may be associated with immunosuppression and neurological impairment in this animal. jaguarundi # died presenting weight loss and gastrointestinal disorders including vomiting, anorexia and diarrhea. histopathology revealed splenic alterations and evidence of enteritis (table ) . although these conditions are commonly associated with felv in domestic cats, it was not possible to ascertain whether they were caused by felv infection in this case. two captive-born jaguarundis, # and # , presented test results similar to those reported for domestic cats with abortive felv infection and seroconversion as the only marker of felv exposure [ ] . the female jaguarundi # had a history of direct contact with the two jaguarundis with progressive felv infection, # and # : she was a sibling of jaguarundi # and had mated with the male # . relationships at the protein level. the most parsimonious tree, with length = , is shown. the consistency index is ( . ), the retention index is ( . ), and the composite index is . ( . ) for all sites and parsimony-informative sites (in parentheses). there were a total of positions in the final dataset. the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test ( replicates) are shown next to the branches. the tree is drawn to scale, with the length being relative to the number of changes over the entire sequence. the mp tree was obtained using the subtree-pruning-regrafting (spr) algorithm with search level in which the initial trees were obtained by the random addition of sequences ( replicates). all positions containing gaps and missing data were eliminated. analyses were conducted in mega [ ] [ ] [ ] [ ] ] this would explain how she was exposed to felv, either at the same time as her sibling # or during mating with # . subsequently, jaguarundi # gave birth to jaguarundi # , who did not show any signs of felv exposure (tables , , ). this may well be the case, since the mother, # , had developed an antibody response and probably had never shed felv (abortive infection). it is unknown for how long and at what virus challenge conditions jaguarundi # was exposed, as animals were naturally and not experimentally infected in the present study. overall, we speculate that jaguarundi # developed an abortive felv infection for similar (and possibly several) reasons that may drive felv abortive infections in some domestic cats. the parents of male jaguarundi # were the felv-negative male jaguarundi # and the female # . thus, it is unknown at what time point jaguarundi # was exposed to felv. overall, we provide evidence of progressive and abortive felv infection in p. yagouaroundi. similar results have been found in florida panthers (puma concolor coryi), which belong to the felid lineage (genus puma), the same phylogenetic lineage as the jaguarundis [ ] . in florida panthers, infection outcomes resembled those of domestic cats with progressive, regressive and abortive infection (previously persistent, regressive, and latent infection) [ , ] . we speculate that felv infection in jaguarundis is similarly unpredictable, and it is influenced for the same diverse aspects cited above for domestic cats. complimentary to the fact that felv causes various tumors in domestic cats [ ] , the literature has reported a felv-associated multicentric t-cell lymphoma in a captive non-domestic cheetah (acinonyx jubatus) [ ] and non-felv-associated t-and b-cell lymphomas in geriatric african lions (panthera leo) [ ] . these data motivated the search for possible felv involvement in the appearance of the neoplastic intra-abdominal mass in jaguarundi # . although jaguarundi # had not shown any evidence of felv infection intra vitam (table ) , we further investigated whether felv antigens or proviruses were present in intestinal lymphoid cells and whether these cells produced felv viral rna locally without it entering the bloodstreamas would be expected in a case of sequestered felv infection [ ] . however, no felv antigens were detected by immunohistochemistry and no felv provirus or viral rna were detected by molecular assays. infection with fiv, which is a lentivirus associated with lymphomagenesis in domestic cats [ ] , was not detected by the commercial immunoassay, and only antibodies specific to fiv p capsid proteins were detected by western blot, which may be indicative of an early or very late fiv infection or may have resulted from unspecific cross-reactivity. the fiv rt-pcr assays performed from whole blood were negative; however, this may be due to low viral loads or lack of specificity of the assays due to sequence diversity of different fiv isolates. in conclusion, jaguarundi # had developed a non felv-associated intestinal b-cell lymphoma; involvement of fiv in the development of the neoplasia cannot completely be ruled out. notably, a spleen sample from jaguarundi # had been previously analyzed for the presence of the felv receptor fthtr , which permits the virus to enter the cell. the fthtr complementary dna (cdna) from that animal had shown % nucleotide and amino acid identity to the fthtr cdna sequences of the domestic cat and other wild felid species, such as the lynx (lynx pardinus), african lion (p. leo bleyenberghi), asiatic lion (p. leo persica), and european wildcat (f. silvestris silvestris) [ ] . in addition, fthtr was quantified by real-time pcr in a few tissues from two jaguarundis where tissue was available (the viremic jaguarundi # and jaguarundi # , which developed a tumor, but was not felv-infected).these limited results showed no significant difference between the fthtr tissue loads in cat and jaguarundi tissue (results not shown). however, it also needs to be mentioned that the real-time pcr assay was designed for the domestic cat thtr and not the jaguarundi thtr . two-point mutations can be found in the region of the assay: one-point mutation in the middle of the forward primer and one in the probe. thus, although similar fthtr expression levels were found in jaguarundi tissue compared with cat tissue, we cannot exclude differences in the efficiency of the real-time pcr assay. these findings support the perspective that the first phase of the felv virus cycleviral entrymight be similar among domestic cats and other wild felid species. we did not detect enfelv in the jaguarundis by qpcr. this is consistent with earlier data [ ] in which enfelv was not detected in jaguarundis. accordingly, felv-b, which has greater pathogenicity than felv-a in domestic cats and arises by recombination of exogenous felv-a with enfelv sequences [ ] , was not found in the two felv-positive jaguarundis # and # . moreover, the highly virulent felv-c was not detected. in felvinfected iberian lynxes, enfelv sequences were also not detected, but their felv-a variants were shown to be highly virulent, suggesting that the mechanisms inducing disease in these wild felids might be distinct from those in domestic cats [ ] . the env sequence obtained from the felv infections of jaguarundis # and # [kr ] clustered with the highest identity with the felv-faids and felv- strains, which represent members of a highly conserved group of horizontally transmitted, minimally pathogenic felv-a present in all naturally occurring infections in domestic cats. both felv strains were originally isolated from cats in the united states of america (usa) [ , ] . interestingly, the surface unit of the env sequence of felv detected in the jaguarundis also showed % identity to those of the env gene of felv isolated from the critically endangered iberian lynxes [eu to eu ] in spain, whose small wild population suffered from an outbreak of felv with several deaths [ ] . both pcr-positive jaguarundis, # and # , were infected with a felv with an identical env sequence, indicating a common origin of the virus. it is unknown at what time point each of the jaguarundis became infected. the reproductive success achieved for jaguarundis in captivity is probably a result of the breeding program conceived for small felids at fpzsp [ ] and a number of measures for improving the well-being of the animals, including the location of the jaguarundi enclosures in an isolated and forested area of the zoo away from the public. nonetheless, domestic cats commonly are seen invading the zoo, especially the more isolated areas, and they were probably the source of infection of felv for the jaguarundis. domestic cats are the main reservoir for felv worldwide and greatly outnumber felv-infected wild felids. given this, it is prudent to prevent the presence of domestic cats in nature reserves, zoos and other settings with captive wild felids and to reinforce the importance of control measures for felv in domestic cats, such as testing and vaccination. a severe felv outbreak occurred in a previously naïve population of florida panthers (puma concolor coryi) in north america from to in which five felv antigen-positive panthers died [ , ] . during a six-month period in , six provirus-positive antigenemic iberian lynxes (lynx pardinus) died in spain [ , ] . in both situations, felv vaccination programs were initiated. we recommend safe recombinant subunits or inactivated felv vaccines for captive jaguarundis, especially considering that invading domestic cats, potential sources of infection, are a frequent problem faced by zoos. in addition, we recommend the inclusion of felv testing in breeding programs for jaguarundis. our findings support our hypothesis that felv infection in p. yagouaroundi mimics the outcomes observed for the domestic cat. in addition, we found phylogenetic evidence that domestic cats may have been the source of the felv-a infection of the jaguarundis. despite the minimal pathogenicity of felv-a for domestic cats, this virus may represent a threat to the jaguarundi population, and regular felv testing and vaccination are encouraged. herpailurus yagouaroundi. in: the iucn red list of threatened species 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feline immunodeficiency and feline leukemia virus infection multicentric t-cell lymphoma associated with feline leukemia virus infection in a captive namibian cheetah (acinonyx jubatus) malignant lymphoma in african lions (panthera leo) role of feline immunodeficiency virus in lymphomagenesis -going alone or colluding? quantification and molecular characterization of the feline leukemia virus a receptor genomically intact endogenous feline leukemia viruses of recent origin naturally occurring feline leukemia virus subgroup a and b infections in urban domestic cats experimental transmission and pathogenesis of immunodeficiency syndrome in cats strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses small felid breeding project at sao paulo zoo subtle mutational changes in the su protein of a natural feline leukemia virus subgroup a isolate alter disease spectrum vaccination of cats experimentally infected with feline immunodeficiency virus, using a recombinant feline leukemia virus vaccine recombinant felv vaccine: long-term protection and effect on course and outcome of fiv infection monoclonal antibodies to three epitopic regions of feline leukemia virus p and their use in enzyme-linked immunosorbent assay of p we are deeply indebted to all veterinarians and staff of fundação parque this work was supported by grants of coordination for the improvement of higher education personnel (capes) and the post-graduate pro-rector from usp in brazil and the international relations office from the university of zurich in switzerland. partial writing of this manuscript work was also supported by the são paulo research foundation (fapesp) (grant / - ). the funding bodies had no specific role in the design of the study, collection, analysis, and interpretation of data. the datasets supporting the conclusions of this article are included within the article.authors' contributions cf, jlcd and rhl concepted and designed the study. cf and mcm handled, sampled, and retrieved data from jaguarundis at zoo. cf and khh conducted the serological and molecular tests. cf, jlcd, lnt and mr performed the histopathological and immunohistopathological analyses. khh conducted the sequencing and phylogenetic analyses. all of the authors drafted and approved this version of the manuscript. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -bpnak ov authors: ma, fen-lian; li, dan-di; wei, tian-li; li, jin-song; zheng, li-shu title: quantitative detection of human malawi polyomavirus in nasopharyngeal aspirates, sera, and feces in beijing, china, using real-time taqman-based pcr date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: bpnak ov background: human malawi polyomavirus (mwpyv) was discovered in , but its prevalence and clinical characteristics are largely unknown. methods: we used real-time taqman-based pcr to detect mwpyv in the feces (n = ) of children with diarrhea, nasopharyngeal aspirates (n = ) from children with respiratory infections, and sera (n = ) from healthy adults, and analyzed its clinical characteristics statistically. all the mwpyv-positive specimens were also screened for other common respiratory viruses. results: sixteen specimens were positive for mwpyv, including ( . %) respiratory samples and three ( . %) fecal samples. the samples were all co-infected with other respiratory viruses, most commonly with influenza viruses ( . %) and human coronaviruses ( . %). the mwpyv-positive children were diagnosed with bronchopneumonia or viral diarrhea. they ranged in age from days to years, and the most frequent symptoms were cough and fever. conclusions: real-time pcr is an effective tool for the detection of mwpyv in different types of samples. mwpyv infection mainly occurs in young children, and fecal–oral transmission is a possible route of its transmission. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the family polyomaviridae contains small encapsidated dna viruses with a closed circular dna genome of kb, packaged within an icosahedral capsid structure. the regulatory proteins, large t and small t antigens, are encoded in the early region of the genome, and structural proteins vp , vp , and vp are encoded in the late region [ ] . human polyomaviruses (hpyvs) usually cause asymptomatic primary infections, and persist in the body throughout life [ ] . thirteen hpyvs have been identified and successively isolated from urine (bkpyv), brain tissue (jcpyv), respiratory secretions (kipyv and wupyv), skin (mcpyv, hpyv , hpyv , and tspyv), serum (hpyv ), stool samples (hpyv and isolates mw and mx, stlpyv), liver tissue (hpyv ), and vascular endothelial cells (njpyv). the detection of hpyvs in these tissues suggests there exists an unrecognized tropism for hpyv [ ] . hpyv is the type species of the genus deltapolyomavirus in the family polyomaviridae. in , human polyomavirus (hpyv ), mw polyomavirus (mwpyv), and mx polyomavirus (mxpyv) were isolated from condylomas on the buttocks of patients, fecal specimens from healthy children, and stool samples from children suffering diarrhea, respectively [ ] [ ] [ ] . the whole-genome sequences of mxpyv, mwpyv, and hpyv are nearly identical, indicating that they are variants of the same species, which suggests that hpyv is a fecal contaminant rather than a wart-causing skin virus [ ] . hpyv has not yet been definitively linked to a particular human disease [ ] . very little is known about mwpyv, so it is important to investigate its prevalence, basic epidemiology, genomic variability, and possible pathogenicity. unlike conventional pcr-based approaches, real-time quantitative pcr (qpcr) uses a fully automated detection system, with high sensitivity. different chemistries are commercially available for qpcr, including sybr green, taqman, scorpion, and molecular beacons, but taqman is most frequently used because it can discriminate sequences that differ by only one nucleotide [ ] . in a previous study, we demonstrated the specificity, sensitivity, and reproducibility of a taqman-based real-time qpcr for detecting hpyv [ ] . therefore, in this study, we used realtime qpcr and dna sequencing to investigate the presence of mwpyv in fecal samples from children with diarrhea, nasopharyngeal aspirate (npa) samples from children with acute respiratory tract infections (aris), and sera from healthy adults in china. npa samples were collected from hospitalized children with aris, who ranged in age from days to years, at beijing friendship hospital, china, between october and september . a total of fecal samples were collected from children < years old presenting with viral diarrhea at the capital institute of pediatrics, china, in . the sera of healthy adults, from the institute of blood transfusion, chinese academy medical sciences, were collected between october and june . all specimens were collected and transported immediately to the national institute for viral disease control and prevention, china cdc, and stored at − °c until processing. fecal samples were diluted approximately : in phosphate-buffered saline and filtered through membranes with a pore size of . μm before the dna was extracted. the total nucleic acids were extracted from μl of the fecal, serum, and npa samples with a qiaamp minelute virus spin kit (qiagen, beijing, china; catalogue no. ) and eluted in a volume of μl, according to the manufacturer's instructions. the dna concentrations were determined with a biopho-tometer™ plus (eppendorf, germany) at a wavelength of nm (a ), and dna purity was evaluated with the a /a ratio. the samples were stored at − °c for subsequent use. a taqman real-time qpcr assay was designed to target the mwpyv vp gene using the real time pcr tool (integrated dna technologies, inc.). the binding sites for the primers and probes used for mwpyv share % sequence identity with the vp sequences reported for hpyv and mxpyv. the primers and probe used for this assay were ′-ggagaccccactttaactagac- ′ (forward primer), ′-acttggcatatctgttactg gg- ′ (reverse primer), and ′-fam-atatggattt gcgttgctgcccg-tamra- ′ (taqman probe, where fam is tamra). the resulting amplicon was bp in length. the qpcr was performed in a total volume of μl containing μl of × taqman gene expression master mix (takara, japan), . μl of each primer ( pmol/μl), . μl of probe ( pmol/μl), . μl of diethyl-pyrocarbonate-treated water, and μl of pmd -t/vp plasmid (plasmid pmd -t linked to the -bp target sequence). the samples were tested in a -wellplate format, with sterile water as the negative control. the cycling conditions were °c for min, °c for min, followed by cycles of °c for s and °c for min, using the mx p qpcr system (agilent stratagene, usa). serial -fold dilutions of pmd -vp were prepared, with the standard plasmid serially diluted from to copies/μl. the dilutions were added to the real-time qpcr reactions, and analyzed in triplicate. the results were used to construct a standard curve. the specificity of the qpcr was assessed by also testing wupyv, kipyv, jcpyv, bkpyv, and hpyv . reproducibility was evaluated by adding - copies/μl pmd -t/ vp to each reaction, and performing all the reactions four times, with the housekeeping gene glyceraldehyd- phosphate dehydrogenase as the internal control. the nucleic acid ( μl) from each npa, fecal, or serum sample was added to a real-time qpcr reaction. the ct values were measured as the point at which the fluorescent signal of the sample crossed the threshold value. all the mwpyv-positive npa specimens were also screened for influenza a virus and influenza b virus, parainfluenza viruses (pivs) - , human metapneumovirus (hmpv), respiratory syncytial virus (rsv) and human coronaviruses (hcos) nl , oc , e, and hku , using the agpath-id™ one-step rt-pcr reagent (invitrogen, life technologies inc.), and for human bocavirus (hbov) using taqman real-time qpcr. the agpath-id™ one-step rt-pcr cycling program was: °c for min, °c for min, followed by cycles of °c for s and °c for s. a ct value of ≤ was confirmed with dna sequencing. all qpcr-positive samples were tested with conventional pcr using the primers described. the amplified pcr products were cloned into the pmd -t vector (takara) for sequencing (invitrogen, life technologies inc.). the complete vp gene of mwpyv was amplified with nested pcr in a μl reaction volume that contained . μl of × ex taq buffer (takara), μl of forward primer ( pmol/μl), μl of reverse primer ( pmol/μl), μl of dntp mix ( . mmol/l), . μl of ex taq dna polymerase (takara), μl of positive-sample nucleic acid, and μl of sterile water. the primers used in this study are listed in additional file : table s . the amplified products were cloned into pmd -t for sequencing. the full-length vp sequence was aligned with the sequences of hpyvs available in genbank with the nucleotide blast program. the sequences have been submitted to the genbank database. clinical data, including the demographic data (age and sex), and the vital symptoms and signs at admission, were considered. statistical comparisons were made with fisher's two-tailed exact test and the mann-whitney u test. all analyses were performed with spss . software. the sequences determined in this study were submitted to genbank under accession numbers ky , ky , ky and ky . to determine the analytical sensitivity, different concentrations of the plasmid pmd -vp , ranging from to copies/μl, were used. the slope of the standard curve was − . and r = . , the efficiency was . and the limit of detection was copies. these parameters are suitable for quantifying the mwpyv load. the assay did not amplify any product from the other viral pathogens tested (wupyv, kipyv, jcpyv, bkpyv, and hpyv ), indicating that the primers and probe are specific for the detection of mwpyv. four different dna concentrations ( - copies per reaction) were each tested four times in each run. the maximum coefficient of variation was . %, indicating good precision (data not shown). mwpyv was only detected in respiratory and fecal specimens. with real-time qpcr, ( . %) of the npa specimens were positive for mwpyv; three ( . %) of the fecal samples were positive for mwpyv; and no mwpyv was detected in the serum specimens. the screening results for the various sample types are presented in table . of the children with aris included in the study, were male, so the male:female ratio was . : . the age range was days to years. the mwpyv prevalence rate was . % ( / ), and was higher in males ( / , . %) than in females ( / , . %). the infected children ranged in age from days to years, and children aged ≤ years accounted for . % ( / ) of the total mwpyv-positive children. children aged - months had the highest infection rate ( / , . %), and the infection rate of those aged - months ranked second ( / , . %) (fig. a) . mwpyv was detected throughout the year, except in january, april, july, and november. the mwpyv infections were mainly sporadic: one case in october , one case in december , two cases in february , one case in march , three cases in may , two cases in june , two cases in august , and one case in september (fig. b) . the peak incidence ( / , . %) occurred in october . the majority of fecal samples were collected in , with approximately equal numbers of samples collected each month in the first half of the year, except that - samples were collected per month from july to october. of the children with diarrhea included in the study, were males, so the male:female ratio was . : . the age range was days to . years, and children aged . - months accounted for . % ( / ), and those aged - months accounted for . % ( / ). the prevalence of mwpyv was . % ( / ). of the three positive patients, more were male ( / , . %) than female ( / , . %). the two infected male children were and months old, and the female was months old, so all three mwpyv-positive patients were < years. these mwpyv infections occurred in january, february, and june. of the mwpyv-positive npa samples, ( %) were co-infected, eight with one other respiratory virus and four with two other respiratory viruses. influenza virus was detected most frequently ( / , . %), including influenza a virus ( . %) and influenza b virus ( . %), followed by human coronaviruses (hco, . %: hco-oc , %; hco-nl , . %), human parainfluenza virus type (piv , . %), and human bocavirus (hbov, . %). one of the samples (sample bj , . %) was co-infected with rotavirus. all the fecal specimens had already tested positive for rotavirus, and / (fecal samples bj and bj , . %) were also coinfected with influenza a virus ( table ). no evidence indicated that the illnesses of the co-infected patients were more serious than those of patients with single mwpyv infections (results not shown). mwpyv-positive children with aris were diagnosed with bronchopneumonia ( / , . %), newborn pneumonia, using the standard curve method, we calculated the viral genome copy numbers in the positive npa specimens to be . - . copies/μl based on the previously established real-time pcr assay ( table ). the genome copy number was . copies/μl in one child with both bronchopneumonia and rotaviral enteritis, which was much higher than the . copies/μl npa in children suffering bronchopneumonia and other diseases (p < . , mann-whitney u test). the clinical symptoms of all three mwpyv-positive children with diarrhea included diarrhea and fever ( %). the viral genome copy numbers in the three positive fecal specimens, calculated with the standard curve, were . , . , and . copies/μl. the sequences of the -bp pcr products from all the mwpyv-positive npa and fecal samples were % identical to the corresponding mwpyv, mxpyv, and hpyv sequences, according to a blastn comparison. to determine the complete genomic sequence of the vp gene, four genomic fragments were amplified with nested pcr using specific primers. the complete vp genomic sequences of only four mwpyv-positive npa samples (bj , bj , bj , and bj ) were amplified. dna sequencing confirmed that these four positive samples contained mwpyv. the bj npa sample contained an mwpyv isolate that shared % sequence identity with strain ma . the mwpyv isolates in samples bj , bj , and bj shared % sequence similarity ( - single-base mutations) with isolates hn , hb c, hb c, and hb c. several studies have reported the widespread distribution of mwpyv in different areas of the world, including africa, the usa, asia, italy, australia, and south america [ , [ ] [ ] [ ] [ ] [ ] . in this study, we used a qpcr assay to rapidly and accurately screen for mwpyv in npa, serum, and fecal samples collected from different populations in china. thirteen ( . %) of the samples from children with aris and three ( . %) from children with diarrhea were positive for mwpyv, whereas no serum (n = ) was positive for mwpyv. in brief, the analysis of clinical samples only detected mwpyv in respiratory and fecal specimens from children, suggesting that the establishment of the primary infection occurs at an early age, and that the gastrointestinal and respiratory tracts are sites of viral persistence. this indicates a possible fecal-oral route of transmission during early childhood. to verify the specificity of the primers and probe used in this study, other viruses (wupyv, kipyv, jcpyv, bkpyv, and hpyv ) were also tested, but no fluorescent signal was detected. we excluded any false-positive results by sequencing all the pcr products. it is noteworthy that all the -bp pcr products from positive samples shared % identity with the corresponding genomic regions of mwpyv, hpyv , and mxpyv, accessed from genbank. the sequences of the complete vp genes confirmed that npa samples bj , bj , bj , and bj contained mwpyv, and revealed that the viruses in the last three samples contained - single-base mutations. in total, mwpyv-positive samples were detected in this study, and the cycle threshold (ct) values for most of these were high (average ct = . , range . - . ), suggesting that the viral load was low. it is possible that the pcr failed to amplify the complete vp gene sequences from the other positive samples. most aris are caused by viruses [ ] . in this study, the prevalence of mwpyv in npa was . %. this is similar to findings in australia ( . %) [ ] , but differs from a study in mexico, which reported a detection rate of . % in nasal washes from children suffering ari [ ] . in a study by rockett et al., mwpyv was the most prevalent polyomavirus ( %) detected in respiratory specimens from children with ari. these findings are supported by other studies that frequently detected mwpyv in respiratory samples from acutely ill patients and babies with upper respiratory symptoms [ , ] . possible explanations for these discrepancies may be the use of different detection assays, differently defined cutoff values, different geographic regions examined, and the varying characteristics of the study populations. mxpyv, mwpyv, and hpyv are presumably human tropic [ ] , and appear to be largely confined to the gastrointestinal tract [ ] . for this reason, we tested fecal specimens from children with a diagnosis of viral diarrhea. we detected mwpyv in . % of fecal specimens from these children, which is lower than previously reported rates of . % and . % in children with diarrhea in st louis, usa, and australia [ , ] . these discrepancies are probably attributable to the different periods at which the samples were collected and the different detection methods used. the detection of mwpyv in the feces of children with diarrhea indicated that mwpyv may not be a strong candidate for gastroenteritis in children because of the coinfection with rotavirus.. wupyv, kipyv, sv , bkv, jcv, mxpyv, and mcv have also been detected in human feces [ , [ ] [ ] [ ] [ ] , although their primary sites of pathology are elsewhere in the human body. seroepidemiology plays an important role in establishing the link between hpyvs and disease and in understanding the dynamics of infection. in this study, mwpyv was undetectable in the blood of healthy adults, consistent with other published data, and mwpyv was not previously detected in the blood of autoimmune children [ ] , as has also been observed for the mxpyv, jcpyv, and bkpyv [ ] . however, in contrast to our findings, the seroprevalence of mwpyv in adulthood was % in a large sample from italy [ ] , % in a sample from colorado [ ] , % in serum samples from the czech republic [ ] , and almost % in samples from new hampshire, usa [ ] . these contrasting data may be attributable to the relatively small number of blood samples we analyzed (n = ) or a different mwpyv may have been isolated from the human samples, to which have affected the antibody binding during serological analysis. moreover, mwpyv dna is more likely to be detected in blood during the primary infection than during viral reactivation [ ] . in a study by nicol et al., mwpyv was cleared or the virus was not reactivated with age [ ] , as has been reported for tspyv [ ] , which may also explain why we detected no mwpyv-positive sera. factors such as age, sex, the time of year when multiple viruses circulate, and a history of immunosuppression are associated with an increased chance of viral co-infection [ ] . in this study, of the mwpyv-positive patients were co-infected with other viruses, and four of these had multiple infections. the primers and probes used in this study are listed in additional file : table s [ ] [ ] [ ] . influenza a virus and hcov were the respiratory viruses most frequently detected in the mwpyv-positive patients, which is consistent with their frequent detection with wupyv and kipyv in respiratory samples [ ] . the detection rate of influenza a virus was . %, followed by hco-oc , and then piv , the predominant subtype among pivs. a comparison of the clinical symptoms experienced by the patients with and without co-infections indicated that co-infection with mwpyv did not affect the severity of the illness. mwpyv-positive children with aris were diagnosed with bronchopneumonia, and their commonest symptoms were fever and cough. the symptoms of the three mwpyv-positive children with diarrhea who were co-infected with rotavirus included diarrhea and fever. our data suggest that primary mwpyv infections occur in childhood, predominantly sporadically. however, it is possible that mwpyv is shed within both the gastrointestinal and respiratory tracts. understanding these factors will help us prevent the transmission of mwpyv infections. to our knowledge, this is the first report of the epidemiological and clinical profiles of mwpyv in children hospitalized with aris or diarrhea in china, based on a real-time qpcr specific for mwpyv. real-time pcr is an effective tool for the detection of mwpyv in different types of samples. mwpyv infection mainly occurs in young children, and fecal-oral transmission is a possible route of its transmission. human polyoma jc virus minor capsid proteins, vp and vp , enhance large t antigen binding to the origin of viral dna replication: evidence for their involvement in regulation of the viral dna replication the role of polyomaviruses in human disease identification of a novel human polyomavirus in organs of the gastrointestinal tract complete genome sequence of a tenth human polyomavirus identification of mw polyomavirus, a novel polyomavirus in human stool discovery of a novel polyomavirus in acute diarrheal samples from children different serologic behavior of mcpyv, tspyv, hpyv , hpyv and hpyv polyomaviruses found on the skin the rapidly expanding family of human polyomaviruses: recent developments in understanding their life cycle and role in human pathology frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in beijing mw polyomavirus and stl polyomavirus present in tonsillar tissues from children with chronic tonsillar disease high diversity of human polyomaviruses in environmental and clinical samples in argentina: detection of jc, bk, merkel-cell, malawi, and human and polyomaviruses acquisition of human polyomaviruses in the first months of life malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors detection of malawi polyomavirus sequences in secondary lymphoid tissues from italian healthy children: a transient site of infection viral etiology of acute lower respiratory tract infections in hospitalized young children in a children's referral hospital in iran detection of novel polyomaviruses, tspyv, hpyv , hpyv , hpyv and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid quantitative detection of merkel cell virus in human tissues and possible mode of transmission polyomavirus shedding in the stool of healthy adults excretion of the novel polyomaviruses ki and wu in the stool of patients with hematological disorders wu polyomavirus in fecal specimens of children with acute gastroenteritis seroprevalence of human malawi polyomavirus seroprevalence rates of hpyv , hpyv , tspyv, hpyv , mwpyv and kipyv polyomaviruses among the healthy blood donors seroepidemiology of human polyomaviruses in a us population the dynamics of herpesvirus and polyomavirus reactivation and shedding in healthy adults: a -month longitudinal study age-specific seroprevalences of merkel cell polyomavirus, human polyomaviruses , , and , and trichodysplasia spinulosa-associated polyomavirus polymicrobial acute respiratory infections in a hospital-based pediatric population field evaluation of taqman array card (tac) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection impact of human coronavirus infections in otherwise healthy children who attended an emergency department human bocavirus in patients with respiratory tract infection prevalence of human ki and wu polyomaviruses in children with acute respiratory tract infection in china we thank the beijing friendship hospital, china, capital institute of pediatrics, china and institute of blood transfusion, chinese academy medical sciences, for providing the samples, and the laboratory staff who contributed to this study. the study was funded by the national major science & technology project for the control and prevention of major infectious diseases of china ( zx ). additional file : table s . primers and probes used to detect respiratory viruses. the datasets supporting the conclusions of this article are included within the article and its additional files.authors' contributions fm and lz conceived, designed and performed the experiments. fm, tw, dl and lz analyzed the data. dl, tw and jl contributed reagents/materials/ analysis tools. fm and lz wrote the manuscript. all authors read and approved the final manuscript. written informed consent for specimen collection, testing and publication was obtained from the patients' parents or guardians. the authors declare that they have no competinginterest.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -w hemznv authors: jans, jop; elmoussaoui, hicham; de groot, ronald; de jonge, marien i.; ferwerda, gerben title: actin- and clathrin-dependent mechanisms regulate interferon gamma release after stimulation of human immune cells with respiratory syncytial virus date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: w hemznv background: respiratory syncytial virus (rsv) can cause recurrent and severe respiratory tract infections. cytoskeletal proteins are often involved during viral infections, either for cell entry or the initiation of the immune response. the importance of actin and clathrin dynamics for cell entry and the initiation of the cellular immune response against rsv in human immune cells is not known yet. the aim of this study was to investigate the role of actin and clathrin on cell entry of rsv and the subsequent effect on t cell activation and interferon gamma release in human immune cells. methods: peripheral blood mononuclear cells and purified monocytes were isolated from healthy adults and stimulated in vitro with rsv. actin and clathrin dynamics were inhibited with respectively cytochalasin d and chlorpromazine. t cell receptor signaling was inhibited with cyclosporin a. flow cytometry was used to determine the role of actin and clathrin on cell entry and t cell activation by rsv. enzyme-linked immunosorbent assays were used to investigate the contribution of actin and clathrin on the release of interferon gamma. results: cell entry, virus gene transcription and interferon gamma release are actin-dependent. post-endocytic processes like the increased expression of major histocompatibility complex ii on monocytes , t cell activation and the release of interferon gamma are clathrin-dependent. finally, t cell receptor signaling affects t cell activation, whereas soluble interleukin is dispensable. conclusion: analysis of cell entry and interferon gamma release after infection with rsv reveals the importance of actin- and clathrin-dependent signaling in human immune cells. insights into the cellular biology of the human immune response against respiratory syncytial virus will provide a better understanding of disease pathogenesis and may prove useful in the development of preventive strategies. respiratory syncytial virus (rsv) is a negative-sense single stranded rna virus of the family paramyxoviridae and is a major burden on the current health care system. in healthy adults, rsv infections are limited to the upper respiratory tract, but remarkably do not generate long-term immunity [ ] . in children and elderly, rsv can cause severe lower respiratory tract infections requiring admission to an intensive care unit in a small percentage of cases. the first line of defense against rsv infection consists of epithelial cells. upon infection, epithelial cells attract antigen-presenting cells, including dendritic cells and monocytes. monocytes and macrophages are able to engulf pathogens leading to antigen-presentation. the monocytic cell is one of the major immune cell types that is susceptible to rsv infection and the role of monocytes and macrophages in the pathogenesis of rsv infections has been appreciated for decades [ ] [ ] [ ] [ ] [ ] [ ] . during rsv infection in mice, the recruitment of monocytes from the bloodstream limits viral replication and reduces disease severity [ ] . viral particles can interact with receptors at the membrane of monocytes resulting in attachment, uptake and initiation of the immune response [ ] [ ] [ ] . under many circumstances, actin or clathrin are essential for receptor-mediated internalization [ ] [ ] [ ] [ ] [ ] . internalization can be regulated differentially dependent on the cell type. uptake of transferrin occurs clathrindependent in macrophages and is not dependent on clathrin in epithelial cells [ ] . cell-specific differences in entry mechanisms between epithelial cells and fibroblasts have been shown for human cytomegalovirus [ ] . previous studies have studied the internalization of rsv in epithelial cells [ ] [ ] [ ] [ ] . no data is available regarding cell entry of rsv in monocytes, which raises the question whether internalization of rsv occurs differentially in innate immune cells. after internalization, immune cells are involved in antigen-presentation, t cell activation and the production of cytokines like interferon gamma (ifn-γ). ifn-γ, a type ii interferon, plays a critical role in the immune response against viral infections [ ] . t cell activation may occur through cytokines like interleukin or through stimulation of the t cell receptor (tcr). the relationship between cell entry, t cell activation and subsequent release of ifn-γ during rsv infection in primary human cells is unknown. peripheral blood mononuclear cells (pbmcs) provide a useful model to investigate the impact of cellular pathways on antiviral immunity. pbmcs contain important cells that reflect the immune response against rsv like dendritic cells, monocytes and t cells [ , [ ] [ ] [ ] [ ] . in this study, we aimed to investigate the regulation of ifn-γ by actin-and clathrin-dependent mechanisms after stimulation of human immune cells with rsv. for this, we used pharmacological inhibitors to inhibit actin and clathrin. hereby, the contribution of actin-and clathrindependent processes on cell entry, t cell activation and induction of ifn-γ in primary human immune cells during rsv infection was studied. we first examined the dynamics of cell entry of rsv into cd + monocytes by using pharmacological inhibitors. cytochalasin d (cytod) and wiskostatin (wisko) have been used in previous literature to inhibit actindependent entry and chlorpromazine (cpz) for clathrindependent entry [ ] [ ] [ ] . a representative figure of the gating strategy to determine internalization of rsv into monocytes is shown (fig. a) . disruption of actin filaments in monocytes with cytod or wisko significantly reduces the internalization of rsv whereas inhibition of clathrin with cpz has no effect on the internalization indicating that cell entry is an actin-dependent process (fig. b) . extracellular binding of rsv on membrane of monocytes occurred and is not reduced by pre-treatment of monocytes with cytod or cpz (fig. c) . to confirm the inhibitory effect of cytod on cell entry, pbmcs were stimulated with rsv for h and virus gene transcription was measured in monocytes. treatment of pbmcs with cytod abrogates virus gene transcription in monocytes whereas cpz has no effect (fig. d) . mean percentages of internalization, binding and virus gene transcription in untreated monocytes were respectively %, % and %. previous literature shows that t cells are the main producers of ifn-γ in our model of pbmcs and stimulation with rsv [ ] . to determine whether the adaptive immune response against rsv is actin-dependent, the ifn-γ release was measured after stimulation of pbmcs with rsv. inhibition of actin with cytod abrogates the rsvinduced ifn-γ release (fig. a) . although cpz has no effect on the internalization of rsv, inhibition of clathrin reduces the release of ifn-γ (fig. b) . both cytod and cpz have no effect on t cell activation after stimulation with the non-specific t cell activator phytohaemagglutinin (pha) (fig. a-b) . from this, we conclude that both cell entry and post-endocytic processes play a role in the adaptive immune response against rsv whereas mere binding of rsv to the cell membrane of monocytes is not sufficient to induce ifn-γ release. to address the discrepancy between clathrin-independent internalization and clathrin-dependent ifn-γ release, we examined whether post-endocytic processes like t cell activation and upregulation of antigen-presenting molecules are clathrin-dependent. cd was used as an activation marker on t cells [ ] . pha was used as positive control for t cell activation. rsv induces activation of cd and cd t cells and pre-treatment of pbmcs with cpz prevents t cell activation after rsv infection ( fig. a-b) . to investigate whether the upregulation of antigen-presenting molecules is inhibited by cpz, the expression levels of mhc-i and mhc-ii on monocytes was determined. rsv induces a significant increase of mhc-i and mhc-ii expression on monocytes ( fig. c-d) . upregulation of mhc-i expression is not affected by treatment of pbmcs with cpz (fig. c) . the rsv-induced upregulation of mhc-ii expression is not present after pre-treatment of pbmcs with cpz indicating that upregulation of mhc-ii is clathrin-dependent (fig. d) . to confirm the role of antigen-presenting molecules and thereby tcr signaling as its ligand, we evaluated whether il- , as a soluble t cell activator, or tcr signaling affects the ifn-γ release after rsv infection. neutralization of il- with il- bp does not inhibit rsv-induced ifn-γ release. as a control, we show that il- bp is able to significantly inhibit candida-induced ifn-γ response, which is consistent with previous literature ( fig. a ) [ ] . the release of ifn-γ is inhibited when tcr signaling is blocked with cyclosporin a (csa) (fig. b ). internalization and subsequent virus gene transcription of rsv in human monocytes and the induction of ifn-γ are actin-dependent. although clathrin is not involved in fig. cell entry and subsequent virus gene transcription of rsv in monocytes is actin-dependent. a gating strategy to determine the amount of internalization of rsv in monocytes. b monocytes were pre-treated with rpmi, cytod ( μg/ml), wisko ( μm) or cpz ( μg/ml). after pre-treatment, monocytes were incubated with rpmi or rsv at moi for h at °c and internalization was measured. c monocytes were pre-treated with rpmi, cytod ( μg/ml), or cpz ( μg/ml). after pre-treatment, monocytes were incubated with rpmi or rsv at moi for h at °c and binding was measured. d pbmcs were pre-treated with rpmi, cytod ( μg/ml) or cpz ( μg/ml). after pre-treatment, pbmcs were incubated with rpmi or rsv at moi for h and virus gene transcription was measured in monocytes. one-way analysis of variance with bonferroni's comparison test was used for statistical analysis. data are normalized to untreated cells and depicted as mean ± sem (n = - ). (**p < . , ***p < . ) (ns = no significant difference between untreated and cytod-treated cells or between untreated and cpz-treated cells) fig. ifn-γ release after rsv infection is actin-and clathrin-dependent. a-b pbmcs were pre-treated with rpmi, cytod ( μg/ml) or cpz ( μg/ml). after pre-treatment, pbmcs were incubated with rpmi, rsv at moi or pha ( μg/ml) for h and ifn-γ release was measured. lower limit of detection: pg/ml. wilcoxon-signed rank test was used for statistical analysis. data are mean ± sem (n = - ). (*p < . ) (nd = non-detectable, ns = no significant difference between untreated and cytod-treated cells or between untreated and cpz-treated cells) the internalization of rsv, upregulation of mhc-ii on monocytes, t cell activation and the release of ifn-γ after rsv infection are clathrin-dependent processes. finally, t cell receptor activation contributes to the rsv-induced ifn-γ response whereas the production of il- is dispensable. actin is required for cell entry of rsv in epithelial cells [ ] . we have shown for the first time, that primary human monocytes also require actin to internalize rsv for subsequent virus gene transcription to occur. actin plays an essential role in processes involving internalization, including receptor-mediated phagocytosis. for the nipah virus, a paramyxovirus like rsv, infection induces the co-internalization of nipah virus receptor ephrinb and, in general, many receptors are internalized in an actin-dependent manner [ ] [ ] [ ] . several receptors are involved in the recognition of rsv at the membrane, like toll-like receptor (tlr ), cd and nucleolin [ , ] . internalization of tlr and cd has been investigated upon bacterial ligand stimulation. in this process, internalization of tlr is dynamin-dependent and cd is actin-dependent [ , ] . clustering of nucleolin is dependent on actin [ ] . whether actin-dependent clustering of nucleolin occurs after rsv infection and whether it is necessary for internalization is unknown. our finding that intact actin filaments are required for internalization of rsv in monocytes narrows the scope of potential receptors that co-occur with cell entry and are required for internalization. besides cell entry, intact actin is required for virus filament formation, viral transmission and the production of cell-associated infectious virus by epithelial cells [ ] [ ] [ ] [ ] . we observed an inhibitory effect of cytod on the release of ifn-γ during an incubation period of h. inhibition of post-endocytic processes like virus filament formation and transmission could therefore explain the observed reduction of ifn-γ after treatment with cytod. however, in our model of pbmcs, monocytes exhibit abortive infection as no replicating virus is detected in the supernatant of pbmcs stimulated with rsv (data not shown). this is in line with the abortive infection of rsv in alveolar macrophages [ ] . therefore, the role of actin on virus filament formation and viral transmission may be applicable to epithelial cells but most likely would not play a role in our model. after inhibition of actin with cytod or wisko, approximately % of the monocytes still remain positive for rsv f protein. permeabilization of the cells in our assay will not exclude extracellular staining of rsv f protein and therefore the remaining rsv-positive monocytes after treatment could reflect extracellular binding of rsv. because cytod does not influence the extracellular binding of rsv, we conclude that actin dynamics are involved in the internalization of rsv by monocytes. actin-independent processes could also explain the incomplete prevention of internalization by cytod. fusion of the f protein of rsv with the cell membrane could result in entry of viral proteins and might act independently of actin filaments. clathrin has previously been implicated for internalization of rsv in an epithelial cell line after an incubation period of h [ ] . contrary, others have shown that internalization of rsv after h is not clathrin-dependent [ ] . the authors conclude that discrepancies between these studies may arise due to a difference in experimental design. the incubation period of h in our study excludes inhibition of postendocytic processes like viral replication and cell-to-cell transmission. in addition, differences between epithelial cells and monocytic cells have been described previously for internalization of nanoparticles and may explain our results compared to epithelial cells [ ] . the role of cellular mechanisms on t cell activation can be studied in our model of pbmcs. besides internalization, inhibition of actin dynamics reduces the ifn-γ response. these data suggest that, at least in part, the intracellular compartment is involved in the induction of an adaptive response and binding of rsv to the outer membrane is not sufficient to induce ifn-γ release. these results are in line with previous data indicating that intact rsv particles are not able to signal via membrane-associated receptors such as tlr [ ] . in our study, inhibition of clathrin has no effect on the internalization of rsv by monocytes, but effectively reduces the induction of ifn-γ. cpz at μg/ml was the highest tolerable concentration without inducing cytoxicity (data not shown). our data suggest that activation of t cells by rsv is clathrin-dependent. calabia-linares et al. observed the importance of clathrin in the formation of the immunological synapse between t cell and antigen-presenting cells [ ] . possibly, inhibition of clathrin reduces the ifn-γ release due to improper formation of the immunological synapse and thereby inefficient t cell activation. less than % of the t cells in the peripheral blood are rsv-specific t cells [ , ] . therefore, the relative high percentage of - % cd + t cells after stimulation with rsv in our experiments most likely indicates that a general activation of t cells occurs. we demonstrate that clathrin plays an essential role in the upregulation of rsv-induced mhc-ii expression on monocytes . the requirement of clathrin for mhc-ii trafficking has been observed previously [ ] . the role of mhc-ii in the induction of ifn-γ is strengthened by the observation that inhibition of tcr signaling, as a ligand for mhc-ii, reduces the ifn-γ response, whereas inhibition of il- has no effect. the combination of reduced t cell activation and mhc-ii expression suggests that clathrin plays a role in the interplay between innate and adaptive immunity against rsv. fig. ifn-γ release after rsv infection is dependent on t-cell receptor signaling. a il- bp was added to pbmcs simultaneously with rpmi, rsv at moi or heat killed candida. ifn-γ release was measured after h. b pbmcs were pre-treated with rpmi or csa ( nm). after pretreatment, pbmcs were incubated with rpmi or rsv at moi for h and ifn-γ release was measured. lower limit of detection: pg/ml. wilcoxon-signed rank test was used for statistical analysis. data are mean ± sem (n = - ). (*p < . ) (nd = non-detectable) (ns = no significant difference between untreated and cpz-treated cells) the simplification of in vivo rsv infections using our model of pbmcs could be a limitation of the study. although others have shown that monocytes can be become infected with rsv and serve as an in vitro model, alveolar macrophages or inflammatory macrophages are the most likely target of infection in vivo [ ] . therefore, there could be differences in the immune response in vivo compared to our model. pre-treatment of isolated monocytes with the inhibitors and performing a subsequent co-culture with autologous t cells could be considered as an alternative instead of simultaneous culture of all cells present in pbmcs. however, during rsv infection in vivo, multiple immune cells, including monocytes, natural killer cells and t cells are recruited to the lung tissue [ ] . our model can give more insights in the interplay between rsv and different immune cells that are present during rsv infection. in conclusion, this study underlines the importance of actin and clathrin dynamics during rsv infections for cell entry and t cell activation. currently, there is no effective treatment or vaccine against rsv infections. understanding the different aspects of rsv disease pathogenesis and immunity, from early virus-cell interactions to final induction of the adaptive immune response may contribute to the development of novel therapies and effective vaccines. the aim of this study was to investigate the role of actin and clathrin on cell entry of rsv and the subsequent effect on t cell activation and release of ifn-γ in human immune cells. cell entry in human monocytes, virus gene transcription and ifn-γ release are actindependent. post-endocytic processes like the increased expression of mhc-ii on monocytes , t cell activation and the release of ifn-γ are clathrin-dependent. finally, t cell receptor signaling affects the release of ifn-γ, whereas soluble interleukin is dispensable. insights into the cellular biology of the human immune response against rsv will provide a better understanding of disease pathogenesis. actin-dependent pathways were inhibited by pre-treatment of cells with cytod ( μg/ml) or wisko ( μm) and clathrin-dependent pathways were inhibited by cpz ( μg/ ml) (sigma-aldrich). csa (sigma-aldrich) and il- bp (r&d systems, united kingdom) were used to respectively inhibit t cell receptor signaling and il- . no cytotoxic effect determined as annexin v+/ -aad+ cells was observed when using the indicated concentrations of the inhibitor (data not shown). after obtaining informed consent, peripheral blood mononuclear cells (pbmcs) from healthy adults were isolated using lymphoprep and isolation of monocytes from the pbmc fraction was performed using an indirect negative selection magnetic labeling kit (monocyte isolation kit ii human; miltenyi biotec). each -well plate was filled with x pbmc per well or x isolated monocytes per well. the study was approved by the committee on research involved human subjects of the radboudumc. rsv a containing an additional transcription unit encoding gfp (rgrsv) was cultured as previously described [ ] . rgrsv was cultured on hela cells (atcc, ccl- ) in dulbecco's minimum essential medium (dmem) with % fetal calf serum (fcs) and % penicillin/streptomycin. near-confluent hela cells were infected with rgrsv and incubated for three days at °c. cells were scraped and the suspension was centrifuged to remove cellular debris. rgrsv was ultracentrifuged on a sucrose cushion for purification and titrated on hela cells. confluent hela cells ( - %) were infected with fivefold viral dilutions for - h. virus titer was determined by counting wells with ≥ and ≤ infected cells/view (ckx microscope; olympus, tokyo, japan). rgrsv was snapfrozen and stored at − °c until use. pbmcs or isolated monocytes were treated with cytod ( μg/ml), wisko ( μm) or cpz ( μg/ml) for min at °c. for quantification of cell entry, monocytes were permeabilized with cytofix/perm (bd biosciences) after incubation with rsv at moi ( x iu/ well) for h at °c. cells were incubated for min on ice with mouse anti-respiratory syncytial virus fusion protein antibody (ab ; abcam). goat anti-mouse igg pe (bd pharmingen) was used as secondary antibody. for the quantification of rsv binding, coincubations of pbmcs with rsv were performed for h at °c and cells were fixed with % paraformaldehyde before staining. pbmcs were treated with cpz ( μg/ml) for min at °c. pbmcs were stimulated with rsv at moi or with pha ( μg/ml) for h. cd expression, as t cell activation marker, was measured with flow cytometry. for gating, cd and cd or cd positive t cells were selected and the percentage of cd positive cells was determined. after pre-treatment with cpz ( μg/ml), pbmcs were incubated with rsv at moi for h and the expression of mhc-i and mhc-ii on monocytes was determined. for gating, cd positive cells were selected and the geometric mean fluorescence intensity (mfi) of mhc-i and mhc-ii was determined. cell surface markers were stained with cd v , cd af , cd v , cd apc-h , cd pe, cd percp-cy . , cd pe-cy , hla-abc pe and hla-dr pe (bd pharmingen). for quantification of rsv, mouse anti-rsv f protein (ab ; abcam) and goat anti-mouse igg pe (bd pharmingen) were used. virus gene transcription in monocytes was determined by gating cd positive cells as monocytes and calculating the percentage of gfp positive cells. cytoxocity was determined by calculating annexin v+/ -aad+ cells (pe annexin v apoptosis detection kit i, bd pharmingen). events were acquired on an lsr ii flow cytometer and analyzed using flowjo. pbmcs were treated with cytod ( μg/ml), cpz ( μg/ ml) or csa ( nm) for min at °c. after pretreatment, cells were stimulated with rsv at moi , pha ( μg/ml) or heat-killed candida albicans ( x / well) for h. il- bp ( μg/ml) was added simultaneously with rsv. concentration of interferon gamma were measured in the cell supernatants by enzymelinked immunosorbent assays (elisa) (sanquin blood supply, the netherlands) with a lower limit of detection of pg/ml. the authors declare that they have no competing interests. author's contributions jj carried out the design of the study, performed the immunoassays and the statistical analyses and drafted the manuscript. he performed the immunoassays and helped draft the manuscript. rdg and mdj participated in the design of the study and helped draft the manuscript. gf carried out the coordination and the design of the study, participated in the statistical analysis and drafted the manuscript. all authors read and approved the final manuscript. immunity to and frequency of reinfection with respiratory syncytial virus respiratory syncytial virus infection of human cord and adult blood monocytes and alveolar macrophages role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro respiratory syncytial virus induced type i ifn 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clathrin and ap- in the trafficking of mhc class ii molecules to antigen-processing compartments antibodies enhance cxcl production during rsv infection of infant and adult immune cells we would like to thank dr. m.e. peeples, ohio state university, for kindly providing us with a transgenic rsv strain expressing renilla-gfp. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -bxijla authors: zhao, zhixun; wu, guohua; zhu, xueliang; yan, xinmin; dou, yongxi; li, jian; zhu, haixia; zhang, qiang; cai, xuepeng title: rna interference targeting virion core protein orf inhibits goatpox virus replication in vero cells date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: bxijla background: goatpox is an economically important disease in goat and sheep-producing areas of the world. many vaccine strategies developed to control the disease are not yet completely successful. hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. however, none of these studies has been attempted to study gtpv. in the interest of exploiting improved methods to control goat pox, it is participated that rnai may provide effective protection against gtpv. in this study we show the suppression of goatpox virus (gtpv) replication via knockdown of virion core protein using rna interference. results: four short interfering rna (sirna) sequences (sirna- , sirna- , sirna- and sirna- ) against a region of gtpv orf were selected. sense and antisense sirna-encoding sequences separated by a hairpin loop sequence were designed as short hairpin rna (shrna) expression cassettes under the control of a human u promoter. orf amplicon was generated using pcr, and then cloned into pegfp-n vector, named as p /egfp. p /egfp and each of the sirna expression cassettes (p , p , p and p ) were co-transfected into bhk- cells. fluorescence detection, flow cytometric analysis, retro transcription pcr (rt-pcr) and real time pcr were used to check the efficiency of rnai. the results showed that the orf -specific sirna- effectively down-regulated the expression of orf . when vero cells were transfected with shrna expression vectors (p /gfp, p /gfp, p /gfp and p /gfp) and then infected with gtpv, gtpv-orf - was found to be the most effective inhibition site in decreasing cytopathic effect (cpe) induced by gtpv. the results presented here indicated that dna-based sirna could effectively inhibit the replication of gtpv (approximately . -fold reduction of viral titers) on vero cells. conclusions: this study demonstrates that vector-based shrna methodology can effectively inhibit gtpv replication on vero cells. simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of gtpv infection. gtpv is a member of the genus capripoxvirus of the family poxviridae [ ] , which also includes the sheeppox virus (sppv) and the lumpy skin disease virus (lsdv) of cattle. both sheeppox and goatpox are endemic in africa, the middle east and many countries in asia, and the diseases caused by these viruses have a significant economic impact on the livestock industry in africa and asia [ ] . gtpv genome is approximately kbp double-stranded dna, which composes at least putative genes, including conserved replicative and structural genes and genes likely involved in virulence and host range [ ] . orf encodes the virion protein which constitutes a great part of the total protein content of the virion and is essential during the assembly and disassembly of virion. it is similar to myxoma virus (myxv) m l (accession no.af ) [ , ] and vaccinia virus (vacv) a l (accession no.m ) that encodes a kda acidic protein, a part of the viral core, and is synthesized at late stages after infection [ , ] . rna-mediated interference (rnai) is a conserved gene-silencing mechanism, where by the doublestranded rna matching is used as a signal to trigger the sequence-specific degradation of homologous mrna [ ] . rnai can be triggered by chemically synthesised and enzymatically produced - nt long rna duplexes in mammalian cells [ , ] . since the effect of short interfering rnas (sirnas) is generally temporal in transfected animal cells, small rna expression vectors have been developed to induce long-lasting rna silencing in mammalian cells [ ] [ ] [ ] [ ] . rnai represents a new antiviral method and is being increasingly used to inhibit the replication of viral pathogens [ ] such as foot-and-mouth disease [ , ] , porcine reproductive and respiratory syndrome virus [ ] , newcastle disease virus [ ] , classical swine fever virus [ ] and monkeypox virus [ ] . hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses [ , [ ] [ ] [ ] [ ] [ ] . this study provides not only an experimental basis for the development of a new anti-gtpv strategy, but also for a new approach to the study of gtpv infection and replication. goatpox virus strain a/goat/qinghai/av / (a celladapted strain) was used in this study and maintained in african green monkey kidney cells (vero). baby hamster kidney cells (bhk- ) and the gtpv permissive cell line vero (lanzhou veterinary research institute, chin) were cultured in dulbecco's modified eagle's medium (dmem; sigma) supplemented with % heat-inactivated fetal bovine serum (fbs; hangzhou, china), u/ml penicillin and μg/ml streptomycin (sigma). cultures were incubated at °c with % co . the cdna cassettes corresponding to the conserved gene of the gtpv genome was cloned into the pegfp-n vector ( figure a ). directed cloning pcr was used to amplify the orf gene, using the following primers (sense: '-gtcctcgagatggacttcat-gaaaaaatatactaa- ' and antisense: '-gcggatccttgctgttattatcatctagtttg- ') used for amplification contained the target sequences for xhoi (ctcgag) and bamhi (ggatcc) incorporated at the ' of the viral complementary sequence. forward primer contained an atg sequence, before the sequence that codified for the protein, as a start initiation codon of protein translation. the reverse primer uncontained a tta sequence, which was used in the characterization of orf protein and egfp coexpression. pcr products were digested with xhoi and bamhi, and cloned into the pegfp-n expression vector (invitrogen, inc., shanghai, china). the final construct p /egfp ( figure a ) was analyzed by restriction digestion and sequenced. plasmids used for sirnas. an inverted repeat is inserted at the '-end of the human u promoter. the forward sequence of the repeat is or nt long, corresponding to the region of interest of the orf gene. the forward and reverse motifs are separated by a -nt spacer, '-tcaagag- '. the transcriptional termination signal of five ts is added at the '-end of the inverted repeat. the synthesized rna is predicted to fold back to form a hairpin dsrna, which would be finally processed into the putative sirnas. transfection were purified with the wizard purefection tm plasmid dna purification system (promega, usa) and quantified by biophotometer (eppendorf, germany). as the aagn uu sequence (n, any nucleotide) has been found to be preferred for sirna-mediated gene silencing under the control of the polu promoter [ ] , we searched for this sequence in the orf of orf gene. four fragments (orf - , orf - , orf - and orf - ) in the coding region of orf gene were selected according to the web-based criteria (http://www.ambion.com). these selected sequences were then submitted to a blast searching against human genome sequence to check whether or not these potential targets have homologues in the human genome was not targeted. to construct hairpin sirna expression cassette, the following dna oligonucleotides were synthesized: gtpv-orf - , gtpv-orf - , gtpv-orf - and gtpv-orf - ( figure a ). the nt target sequences served as a basis for the design of the two complementary - mer sirna template oligonucleotides that were synthesized, annealed, and inserted into bamhi and bbsi sites of the sirna expression vectors pgpu /neo and pgpu /gfp/neo (genepharma co., ltd, shanghai, china), respectively. the recombinant plasmids were designated as p , p /gfp, p , p /gfp, p , p / gfp, p and p /gfp. the pc and pc/gfp negative controls (genepharma co., ltd, shanghai, china) were negative control plasmid, which encode hairpin sirna that does not have homologues in mice, human and carpripoxvirus genome databases. transfection of the sirna expression cassettes into bhk- cells bhk- cells were seeded in six-well plates and cultured at °c and % co overnight. when the cells showed - % confluence, . μg of p , p , p or p each were cotransfected with an equal amount of p / egfp using fugene hd transfection reagent (roche, germany) according to the manufacturer's recommendations, respectively. simultaneously, . μg of p / egfp were cotransfected with . μg of pc. non-transfected bhk- cells were also used as a control. after an additional h of incubation, cells were observed for the expression of green fluorescent protein in the transfected cells was monitoring fluorescent microscope (olympus, japan). cells were further subjected to fluorescence-activated cell sorting (facs). at h posttransfection, the transfected cells and the controls were washed gently in phosphate-buffered saline (pbs), trypsinized and resuspended in pbs. egfp positive cells and egfp expression signal were evaluated by the facs calibur flow cytometry system (becton dickinson, usa). to confirm the efficacy of rnai, rt-pcr was used to amplify the target gene in the transfected cells. total rna was extracted from bhk- culture with trizol reagent (takara, dalian), and incubated for h at °c with dnase rq (takara, dalian). to detect orf mrna expression in bhk- cells, rt-pcr was conducted using . μg of rna extracts with superscript one-step rt-pcr system (gibco, brl). retrotranscription β-actin as a control was also amplified using the primers '-cacccgcgagtacaaccttc- ' (sense) and '-cccatacccaccatcacacc- ' (antisense). pcr was run for cycles with °c for s, °c for s and °c for s. to verify primer specificity, a melting curve was analyzed, and rt-pcr products were further cloned into pmd -t for sequencing. in order to use full-length orf gene as a quantitative rt-pcr standard, selected primer sequences were ctgtctacatgattaacccactcgttcttc (orf fp primer), and gaagtcgactac ccctctccctatcagggtcatc (orf rp primer). one additional primer was synthesized for quantification of the orf in real-time pcr: -fam-ccttgctcgcgaatttctcaccgatamra- (taqman probe). the target region of real time rt-pcr was - bp of orf . for quantitative analysis of the orf gene, ng total rna from p /egfp-transfected cells was mixed with μl orf primer, heated to °c for min and chilled on ice for min. to this primer template mix was added × buffer ( μl), mmol/μl dntp ( μl), rnasin ( μl), amv reverse transcriptase ( μl, promega, usa) and ddh o to a total volume of μl. the reaction mixture was incubated at °c for min, followed by inactivation of reverse transcriptase at °c for min. real-time pcr was performed with the abi prism sequence detection system using μl transcriptase products as template under the conditions of °c for min, followed by cycles of denaturation at °c for s, annealing, and extension at °c for s. the quantitative standard curve for determination of orf copy number was created by real-time pcr of standard plasmid p /egfp serial -fold dilutions of a stock containing copies/μl. the specificity of the real-time pcr was confirmed by sequencing of the product. four sirnas targeting orf were designed to inhibit gtpv orf gene expression in bhk- cells. we used a modified cmv promoter, a typical rna poliii promoter, to drive the transcription of the sirnas. to monitor the effects of the sirnas, eukaryotic expression plasmid p /egfp was constructed, in which the orf gene were fused to the '-end of the egfp coding sequence, and cotransfected with their specific sirna expression plasmids. so the inhibitory effects of the orf -specific sirnas on the orf expression could be indirectly evaluated by the expression of egfp in the transfected cells. to test whether the expressed sirnas inhibited gtpv production, we first assessed the growing capacity of gtpv in vero cells expressing sirnas. vero cells were seeded in six-well plates and cultured at °c and % co overnight. when the cell reached - % confluence, . μg of p /gfp, p /gfp, p /gfp or p / gfp each were cotransfected with an equal amount of pc/gfp as described above, respectively. the nonspecific vector pc/gfp and non-transfected vero cells were also used as a control. h posttransfection, the cells were infected with gtpv at a multiplicity of infection (moi) of about . . briefly, after removing the culture medium, gtpv ( μl) in infection medium ( . μg/ml trypsin), respectively, were added to each well. the cultures were then incubated at °c, % co for h, at which point the culture medium was replaced with fresh dmem containing % fetal bovine serum. to determine transfection efficiency, we monitored gfp fluorescence intensity of transfected cells using fluorescent microscope analysis. culture supernatants were collected for virus titration. six days post infection, supernatants was harvested from the infected cultures and virus titer (tcid ) was determined three times on vero cells. virus infectivity was determined by serial dilutions of the samples in -well plates and the virus titer was calculated as a tcid by the reed-muench method [ ] . a viral suspension titrated at - to - tcid per . ml was used for viral challenge. vero cells (about % confluent) grown in -well plates were transiently transfected with . μg p /gfp, p /gfp, p /gfp and p /gfp, respectively, per well. after h of transfected, the transfection medium was removed and the cells were washed twice with dmem medium. the transfected cells were then infected with tcid of virus per . ml per well. after additional h incubation, the inoculum was removed and the cells were washed twice with dmem medium. the cells were then maintained in dmem medium supplemented with % fetal bovine serum for days. for detecting the therapeutic potential of sirna, in another parallel experiment, transfection was performed h post-infection with the virus. gtpv replication in vero cells was evaluated by virus titer (tcid ). transient cellular transfection and analysis of the targeted gene and egfp expression in bhk- cells different sirnas suppressed the expression of fusion green fluorescent protein in bhk- cells is different. the sirnas targeting to the conserved region of gtpv genome were generated in vitro by human recombinant dicer enzyme, as described in figure b . to identify an effective inhibitory effect of sirnas, the cdna cassettes of these regions were inserted into the ' end of enhanced green fluorescent protein (egfp) gene to construct reporter plasmids ( figure ). the reporter plasmids were used to cotransfect bhk- cells with either the homologous sirnas or the heterologous sirnas. the results showed that the number of egfp-expressing cell was markedly reduced in the sample transfected with homologous sirnas than sample transfected with heterologous sirnas or non-transfected ( figure a ). facs demonstrated that the levels of inhibition mediated by the sirnas were similar among the different experiment groups and significantly higher than the control group (cotransfection with heterologous sirnas or without sirnas). the inhibitory effects of the sirnas on expression of egfp were quantitatively validated by flow cytometry h posttransfection. the extent of egfp down regulation was assessed by the mean fluorescence of the positive cells (lr-values) and the rate of egfp positive cells figure b ). compared with the pc, the lr-values of the egfp positive cells were reduced in the cells transfected with orf sirna-specific expression plasmids p , p , p and p , and were reduced by . %, . %, . %and . %, respectively. to further demonstrate the levels of inhibition, cells were collected h post-transfection and rt-pcr analysis was performed. the level of target rna, as determined by rt-pcr, was also significantly reduced in cells transfected with homologous sirnas ( figure a) . to measure the level of gene suppression accurately, qpcr primers and taqman probes directing to orf were designed. we also designed probes and primers directed toβ-actin sequence (serve as internal reference). when normalized for loading differences using the βactin mrna, the orf message in the cells transfected with p , p , p and p were reduced by %, %, %and % (orf message copies ratios of cells transfected with shrna expression vectors/cells transfected with empty vector)( figure b ). there was no significant inhibition in cells transfected with the empty vector pegfp-n and nonspecific shrna expression vector pc. mrna levels of orf (average orf mrna levels in cells treated with p , p , p , p , pc and empty vector were . , . , . , . , . , respectively) or β-actin suggested that the reductions in orf message did not result from poor transfection, nonspecific inhibition or toxicity, because the average mrna levels of β-actin for experimental cells were not significantly reduced compared to the control cells. in addition, the suppressive effect was found to be gene-specific, because the inhibitory effect of empty vector and nonspecific shrna expression vector pc were negligible. these results suggest that the sirna generated by in vitro transcription effectively and specifically inhibit the expression of gtpv orf conserved regions in bhk- cells. to investigate whether or not knockout of orf relieves cytopathic effect (cpe) induced by gtpv, vero cells were transfected by plasmids expressing orf protein-targeted shrnas (p /gfp, p /gfp, p /gfp and p /gfp), respectively. nonspecific shrna expression vector pc/gfp was transfected in parallel. h post-transfection, the cultures were infected with gtpv and checked daily. six days later, we found that cells pre-transfected with p /gfp exhibited less cpe, whereas other shrna-treated cells and the empty vector control demonstrated the same typical gtpv-induced cpe as cells infected only with virus, as shown in figure . the tcid assay was performed to examine the effect of sirna on production of viable virus, and the results ( figure ) showed that in control cells the titers reached - . tcid / . ml at days post-infection. in contrast, the titers in the cells transfected with p / gfp, p /gfp, p /gfp or p /gfp, were - . , - . , - . and - . / . ml days post-infection rnai is a process of sequence-specific, post-transcriptional gene silencing that is initiated by double stranded rna. introduction of sirna results in degradation of sirna specific transcripts thus reducing the expression of their protein product. in plants, it is a natural antiviral defense mechanism [ ] . in mammalian cells, however, dsrnas longer than nt activate an antiviral defense leading to the nonspecific degradation of rna transcripts and a general shutdown of host-cell protein translation [ ] . the successful use of sirna in animal cells encouraged the development of sirna expression vector [ ] and numerous studies have demonstrated that dna-based sirna is a promising approach for antiviral therapy in mammals. rnai represents a new antiviral method and is being increasingly used to inhibit the replication of viral pathogens [ ] , such as hiv- [ ] , hepatitis c [ ] , influenza [ ] , severe acute respiratory syndrome [ ] and hepatitis e viruses [ ] . this study has demonstrated the use of pgpu /neo or pgpu /gfp/neo vector-based rnai against gtpv, a major pathogen of goats and sheep. four different sirna targeting viral gene orf , one key gene involved in gtpv replication, successfully reduced viral replication. the results showed that the orf -specific sirnas p could effectively down-regulate the expression of orf , while p , p and p displayed weak activity. additionally, expression of the housekeeping gene β-actin was also analyzed by rt-pcr and quantitative real-time pcr, and no significant difference in the expression of β-actin was observed between the sirnas treatment groups and pc treatment groups. different sirna sequences display widely different efficacies with regard to suppression of gene expression, requiring screening of multiple sequences [ ] . in this research, we have selected four target sequences for rna interference by the software applications, "sirna target finder and design tool" available at http://www.ambion. com/. as the orf gene is well conserved in gtpv and orf protein is a virion core protein and assembly protein in gtpv, we selected orf as a target gene. in order to generate shrna expression cassettes quickly and accurately, we employed a pcr-based strategy to clone sirna sequences. in this strategy, sirna sequences were designed as a single primer sequence of which bp complementary to the human u promoter were added. the resulting pcr products are shrna expression cassettes including the human u promoter. shrnas that have been generated from this expression system are efficiently processed by dicer into sirnas. in addition, in this study, we selected pegfp-n vector that contains an egfp gene as report gene and can be transfected into mammalian cells using any standard transfection method. vero cells transfected with p /gfp, p /gfp, p / gfp, p /gfp and pc/gfp were examined for cpe by virus titration. all results demonstrated that sirna- is the most effective one, and result showed that minimum concentration of the construct p /gfp is . μg required to induce maximum inhibition. blasting orf sequence in genbank revealed that there were capripoxvirus isolates containing identical sequence corresponding to orf . in view of the sequences of the orf genes of gtpv strains from the same genotype, they all share high homology ( - %). therefore, orf gene is a good target to suppress gtpv replication by rnai. in conclusion, this study demonstrates that vector-based shrna methodology can effectively inhibit gtpv replication on vero cells. further study will be required to determine whether such treatment protect against gtpv infection in vivo. still, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of gtpv infection. some observations on the occurrence of lumpy skin disease in nigeria control of capripoxvirus infections. vaccine the genomes of sheeppox and goatpox viruses a poxvirus-encoded pyrin domain protein interacts with asc- to inhibit host inflammatory and apoptotic responses to infection poxvirus proteomics and virus-host protein interactions the vaccinia virus h r gene encodes late gene transcription factor : purification, cloning, and overexpression structural and functional studies of a , -mr immunodominant protein of vaccinia virus molecular biology duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells a simple and cost-effective method for producing small interfering rnas with high efficacy a system for stable expression of short interfering rnas in mammalian cells inhibition of nf-kappab mediated inflammation by sirna expressed by recombinant adeno-associated virus van parijs l: a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference specific gene inhibition by adenovirus-mediated expression of small interfering rna interference of porcine reproductive and respiratory syndrome virus replication on marc- cells using dna-based short interfering rnas rna interference against viruses: strike and counterstrike lentviral-mediated rnai to inhibit target gene expression of the porcine integrin av subunit, the fmdv receptor, and against fmdv infection in pk- cells adenovirus-mediated rna interference against foot-andmouth disease virus infection both in vitro and in vivo inhibition of newcastle disease virus replication by rna interference targeting the matrix protein gene in chicken embryo fibroblasts in vitro inhibition of csfv replication by retroviral vector-mediated rna interference inhibition of monkeypox virus replication by rna interference stable suppression of tumorigenicity by virus-mediated rna interference inhibition of hepatitis b virus in mice by rna interference u promoter-driven sirnas with four uridine ' overhangs efficiently suppress targeted gene expression in mammalian cells short hairpin activated gene silencing in mammalian cells effective expression of small interfering rna in human cells analysis of gene function in somatic mammalian cells using small interfering rnas a simple method of estimating fifty percent end points in vivo imaging of cidofovir treatment of cowpox virus infection maintenance of protein synthesis in spite of mrna breakdown in interferon-treated hela cells infected with reovirus development and application of sirna expression vector sirna-directed inhibition of hiv- infection inhibition of hepatitis c virus protein expression by rna interference inhibition of influenza virus production in virus-infected mice by rna interference inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, a/ b, a/ b and s effective inhibition of hepatitis e virus replication in a cells and piglets by rna interference (rnai) targeting rna-dependent rna polymerase approaches for chemically synthesized sirna and vector-mediated rnai rna interference targeting virion core protein orf inhibits goatpox virus replication in vero cells we thank yadong zheng for thoughtful discussions and assistance during manuscript preparation. authors' contributions qz and xc designed research; zz performed research and wrote the paper; gw helped to construct partial plasmids and analyzed data; xz contributed new reagents/analytic tools; xy provided partial plasmids. hz helped to culture cells; jl helped to culture viruses; yx d had a co-ordination role in this work. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -o x nj authors: wu, yu; li, wei; zhou, qingfeng; li, qunhui; xu, zhichao; shen, hanqin; chen, feng title: characterization and pathogenicity of vero cell-attenuated porcine epidemic diarrhea virus ct strain date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: o x nj background: porcine epidemic diarrhea virus (pedv) has caused enormous economic losses to the global pig industry. currently available pedv vaccine strains have limited protective effects against pedv variant strains. methods: in this study, the highly virulent epidemic virus strain ct was serially passaged in vero cells for up to generations (p ). characterization of the different passages revealed that compared with p and p , p had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. results: pathogenicity experiments using p in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal pedv antigen distribution; the piglet survival rate in the p group was %. furthermore, whole-genome sequencing identified amino acid changes in p , which might be responsible for the attenuated virulence of p . conclusions: thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines. porcine epidemic diarrhea (ped) is an acute and highly infectious intestinal disease in pigs; it is caused by porcine epidemic diarrhea virus (pedv), which has caused enormous economic losses to the global pig industry [ , ] . ped is characterized by diarrhea, vomiting, dehydration, anorexia, weight loss, and high mortality in suckling pigs. although ped also appears in summer, it mainly occurs in winter. pedv infection causes different symptoms according to the pigs' ages; however, in piglets, the symptoms of pedv infection are particularly serious, including a high mortality rate [ ] . pedv belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus and is an enveloped virus with a single-stranded, positive-sense rna genome [ ] . the whole pedv genome is approximately kb nucleotides (nts) long and has a ′-cap and ′-polyadenosyl tail; the genome also includes ′-and ′-untranslated regions and at least open reading frames (orf a, orf b, and orf - ) [ , ] . orf a and orf b encode the replicase polyproteins a and ab, respectively, which undergo autoproteolysis by viral proteases to form nonstructural proteins (nspl- ) [ ] , which participate in the basic mechanisms of viral rna transcription and replication. orf - encode four structural proteins [fibrin (s), membrane protein (m), envelope protein (e), and nucleocapsid protein (n)] as well as coprotein orf [ , ] ; these proteins are arranged in the genome in the following order: ′-orf (la/lb)-s-orf -e-m-n- ′ [ ] . in , the pedv strain cv was identified as the cause of the ped outbreak in belgium [ ] . in october , a highly pathogenic pedv was discovered in china, which caused the worst outbreak on record and quickly swept across the country [ , ] . the variant then caused a pandemic in the united states in spring and spread to canada and mexico. in addition, severe ped outbreaks occurred almost simultaneously in many asian and european countries, such as korea, japan, belgium, and france [ , ] . vaccination is considered effective in the prevention of pedv infection on farms [ ] . several attenuated activated and inactivated vaccines for classical pedv strains, such as cv [ ] , dr [ ] , and kpedv- [ ] , have been developed and made commercially available in many countries [ ] ; however, the efficacy of these traditional vaccines against emerging pedv strains is questionable because of the antigenic and genetic differences between the vaccine strains and the prevalent strains [ ] . therefore, there is an urgent need for a new pedv vaccine against new variant strains. in the present study, the ct strain was serially passaged in vero cells. the growth kinetics and biological characteristics of the different passages were determined. in addition, -day-old piglets were used to assess the pathogenicity of these strains. finally, the whole-genome sequences of the different passages were determined. a safe attenuated pedv strain was developed in this study, thereby providing an important basis for the preparation of an attenuated vaccine. the pedv ct strain, which belongs to the g b subgroup in china, was previously isolated by and stored at our laboratory [ ] . vero cells were obtained from the american type culture collection (atcc: ccl- ), regularly cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (invitrogen, australia) and % antibiotics ( u/ml penicillin, μg/ml streptomycin, and μg/ml fungizone®; gibco™, usa), and maintained at °c in a humidified % co incubator. mouse anti pedv s monoclonal antibody and y -labeled goat anti-mouse igg antibody were prepared and stored at our laboratory. vero cells were grown in a t flask and washed thrice with phosphate-buffered saline (pbs) at % confluency. the cells were then incubated with ml of the pedv ct strain diluted : in virus growth medium {dmem supplemented with antibiotics ( u/ml penicillin, μg/ml streptomycin, and . μg/ml trypsin [gibco])} for h at °c in a humidified % co incubator. then, ml of the virus growth medium was added to the t flask, which was monitored daily for cytopathic effects (cpes). when cpes were observed in > % of the vero cells, the flask was subjected to three cycles of freezethawing. the cells and supernatants were mixed by pipetting, aliquoted, and stored at − °c. these harvested cells were then used as seed stock for the next passage. finally, the cells were further passaged under each condition for up to passages [ , ] . immunofluorescence assay (ifa) the vero cells were inoculated with pedv in -well plates at a multiplicity of infection (moi) of . . at h post infection (hpi), the cells were washed thrice with pbs, fixed at room temperature (rt) with % paraformaldehyde for min, and then permeabilized with . % triton x- (solarbio, china) for min. after washing as described previously, the cells were blocked with % bovine serum albumin (solarbio) for min at rt. the mouse anti pedv s monoclonal antibody and y -labeled goat anti-mouse igg antibody were then used as the primary and secondary antibodies, respectively. finally, the cell nuclei were stained with ′, -diamidino- -phenylindole (vectorlabs, usa) for min at rt in the dark. the cells were then washed thrice with pbs and observed under a fluorescence microscope. vero cell monolayers were separately inoculated with pedv p , p , or p or were mock infected at an moi of . in -well plates. the culture supernatants and cell lysates were collected at , , , , and hpi. after one round of freeze-thawing, following the reed-muench method, the cell culture samples were analyzed in -well plates to determine the titration for % tissue culture infectious dose (tcid /ml) [ ] . total rna was extracted from the cell culture samples using an rneasy kit (magen, china). all primers used to amplify pedv genomic fragments were designed and preserved at our laboratory [ ] . reverse transcription and polymerase chain reaction (pcr) were performed using the primescript™ rt-pcr kit (promega, usa) and primestar® gxl dna polymerase, respectively. the ′-and ′-end sequences were determined using a ′and ′-competition kit (takara). the pcr products were cloned into a pmd -t vector using the topo® ta cloning® kit (invitrogen, usa) and sent to sangon biotech (shanghai, china) for sequencing. for each amplicon, more than three independent clones were sequenced to determine the accurate sequence of a specific genomic region. all sequencing results were then spliced and aligned using lasergene seqman and lasergene megalign, respectively. all reference sequences were obtained from genbank and used for sequence alignment and phylogenetic analysis. all phylogenetic trees were constructed using mega . software. twenty-two -day-old crossbred (duroc × landrace × big white) conventional piglets were obtained from wen's foodstuffs group co., ltd. (guangdong, china). the piglets were determined to be free of antibodies against pedv and confirmed to be negative for the major porcine enteric viruses pdcov, pedv, tgev, and prov [ ] via virus-specific rt-pcr of fecal swab samples [ , ] . the piglets were then placed in separate cages and randomly divided into four experimental groups. each group was placed in a separate room (biosafety level ). the piglets in each group were orally inoculated with ml of the virus culture medium ( × tcid ) or dmem. the piglets were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment [ ] . fecal consistency was scored as follows: , solid; , paste; , semi-liquid; , liquid; and , death [ ] . one piglet from each group was euthanized for histopathological examination at days post infection (dpi). the remaining piglets were euthanized at the end of the study ( dpi). fresh fecal swabs were collected daily, diluted with pbs, stored at − °c, and subjected to three freeze-thaw cycles. viral rna was extracted from the supernatant of the fecal swab samples as described previously. the amount of viral rna in the fecal swab samples was determined using taqman real-time rt-qpcr with the following primers: sense, ′-gaattcccaagggc gaaaat- ′; antisense, ′-ttttcgacaaattc cgcatct- ′. a probe targeting the pedv n gene ( ′-fam-cgtagcaggcttgcttcggaccca-bhq- ′) was also employed. the thermal cycling parameters were as follows: °c for s followed by cycles at °c for s and °c for s. the gross intestinal tract of each piglet was photographed at necropsy, and the intestinal tissue was collected and fixed with % paraformaldehyde. the fixed samples were sent to guangzhou saville for histopathology or ihc with pedc s-specific monoclonal antibodies. in each experimental group, statistical significance was measured using one-way analysis of variance. two-sided probability values < . (p < . ) were considered to indicate statistical significance. correlations between the fc scores and fecal pedv rna drop titers were analyzed using spearman's rank correlation [ ] . the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk- - ). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. biological characteristics of pedv p , p , and p our results indicated that syncytial, vacuole, and cell exfoliation were more obvious after infection with p than with p and p (fig. a) . the three different passage strains were confirmed by ifa using a specific monoclonal antibody against the pedv s protein. red signals were observed in the strain-infected vero cells for all the three passage strains but not in the uninfected vero cells at hpi. obviously, the cells infected with p produced more red pedv antigens (fig. b) . in addition, growth kinetics indicated that p had a higher viral titer than the other two strains at all time points, indicating that p had better cell adaptability (fig. c) . fecal swabs were collected from the piglets and scored for diarrhea during the experiment; the clinical signs of the piglets in each group were also recorded. at dpi, severe diarrhea and vomiting were observed in the p group. in contrast, slight diarrhea and vomiting were observed in the p group. interestingly, there were no obvious clinical symptoms in the p and mock groups (fig. a) . regarding the lesion onset times in the groups, lesions appeared at , , and hpi in the p , p , and p groups, respectively. throughout the experiment, a high diarrhea score was maintained in the p group. in contrast, in the p and p groups, the diarrhea scores began to rise at dpi, peaked at dpi, and gradually returned to normal by dpi (fig. b) . differences in pedv rna shedding in the four experimental groups pedv rna shedding was detected in fecal swab samples using rt-qpcr (fig. ) . during the entire experiment, the p group maintained a high level of pedv rna shedding. in the p group, pedv rna shedding appeared at dpi, peaked at dpi, and gradually recovered by dpi. pedv rna shedding was not observed in the p group until the second day after challenge; the viral shedding peaked at dpi and gradually improved after dpi. the entire experiment lasted for days. the health of the piglets in the p and p groups had recovered by dpi. at dpi, one piglet was selected from each group for dissection and sampling. at necropsy, intestinal tissue, particularly the small intestinal tissue, of the the p group piglet exhibited dilatation and transparency (fig. a-d) . in the p group piglet, the intestinal tissue lesions were more obvious and the intestine was filled with yellow fluid and mesenteric hyperemia (fig. d) . in contrast, the p group piglet exhibited only slight changes in the intestinal tract, and the control animal was extremely healthy with no pathological changes (fig. b) . the jejunum and ileum of each piglet were examined via histopathological and ihc analyses. histopathological analysis revealed severe atrophy and shedding of the intestinal villi of the p group piglet (fig. h and l) ; in contrast, in the p group piglet, the intestinal villi were shortened and fused ( fig. f and j) . intestinal villus atrophy was mild in the p group piglet, and the examined control group piglet exhibited normal histopathology ( fig. e and i) . in contrast, ihc revealed that the pedv antigen was dominant in the cytoplasm of some segments of small intestinal villi. in the p and p group piglets, large amounts of the pedv antigen were detected in severe lesions; in contrast, the antigen was hardly detected in the p group piglet (fig. n and r) ; further, no pedv antigen was found in the small intestine of the control group piglet (fig. m and q) . in the p group, one piglet died at dpi and two others died at dpi. in addition, one piglet each died at and dpi, respectively; thus, the survival rate in the p group was %. in the p group, the survival rate was . % because only one piglet died at dpi. no animals died in the p and control groups ( % survival rate for both groups) (fig. ). the genome length of the pedv ct strain was determined to be , nts. previous studies conducted at fig. biological characteristics of porcine epidemic diarrhea virus strains after , , or passages. a cytopathic effects of the three strains hpi at an moi of . . b immunofluorescence detection results for the p , p , and p strains in vero cells infected at an moi of . at hpi. c growth kinetics of the p , p , and p strains in vero cells at an moi of . . cell lysates were sampled at the designated time points and titrated using the titration for % tissue culture infectious dose infectivity assay. the asterisk means significant difference (***p < . , **p < . , and *p < . ). p , pedv after passages; p , pedv after passages; and p , pedv after passages; moi, multiplicity of infection; hpi, hours post infection our laboratory have reported that the pedv ct strain belongs to clade of the g b subgroup (fig. ) . table lists the changes in the amino acids and nts of the different passage strains. p was observed to contain base mutations, resulting in amino acid mutations. the s gene is a determining feature of pedv's virulence and evolution [ ] . amino acid comparison indicated that there were only four amino acid mutations (d a, f r, s r and c g) between p and p . in addition, several nucleotide point mutations resulted in aa substitutions in orf ab (a s, t n, d g, h y, y s and v i), orf (t m), e (p l) and m (g d) proteins. we found that p had base mutations (thus, amino acid mutations). moreover, compared with p , p contained base mutations (thus, amino acid mutations) ( table ). in addition, p had high homology with the cv vaccine strain ( . %) and the attenuated dr strain ( . %). the complete genome sequences of the ct strains described here have been deposited in genbank under accession no.mn . ped outbreaks have caused huge economic losses to the pig industry in not only china but also the united states, japan, south korea, and other countries [ , ] . currently, vaccination is an effective solution to tackling the virus; however, owing to variations in the virus [ ] , the classical attenuated vaccine does not provide effective protection. thus, the epidemic strains of the virus must be urgently studied to prepare a new attenuated vaccine. in the classical method, attenuated vaccines are prepared via cell passaging [ , , , ] . some previous studies have reported that increased viral titers and enhanced cell adaptability are the characteristics of viruses with weakened virulence [ , , ] . in the present study, we passaged the epidemic pedv ct strain continuously in vero cells for up to generations and observed that p had better cell adaptability than the other two strains; thus, our findings illustrated that increased passaging increases the cell adaptability and titers. finding balance between cell and animal adaptability and then determining the best generation is the key to the preparation of attenuated vaccine candidate strains via continuous passaging [ , , ] . moreover, the adaptability of the strain to the host animal is characterized by the virulence of the strain, which is usually evaluated via pathogenicity testing of the animal infected with the virus. to prove that . e-h h&e-stained jejunum tissue sections of the different groups at dpi. i-l h&e-stained ileum tissue sections of the different groups at dpi. m-p immunohistochemically stained jejunum tissue sections of the different groups at dpi pedv antigen is indicated by arrows). q-t immunohistochemically stained ileum tissue sections of the different groups at dpi (pedv antigen is indicated by arrows). p , pedv after passages; p , pedv after passages; and p , pedv after passages; h&e, hematoxylin and eosin; pedv, porcine epidemic diarrhea virus fig. survival rates of piglets infected with different passages of porcine epidemic diarrhea virus ct strain. **p < . , and *p < . the strain virulence has been weakened, animal experiments were designed to confirm the strain [ , , ] . thus, the virulence of the three pedv strains was assessed to confirm whether the p strain was weakened. according to data from animal experiments, the p strain could significantly reduce clinical symptoms, viral shedding, and histopathological changes in piglets. therefore, our results illustrated that the p strain had relatively weak virulence and that compared with the p and p strains, it caused lesser damage to the piglets. thus, it can be concluded that the p strain is safe and potentially useful as a candidate strain for the preparation of an attenuated vaccine. to further assess the attenuation of the p strain, we sequenced the whole genomes of the three passage strains. and the method to prepare attenuated vaccine by cell passage can also get clues to the key virulence factors of the virus [ ] . the pedv s protein is involved in receptor binding, viral entry, neutralizing antibody production induction, and host cell fusion [ ] . the s protein of pedv has always been used as a marker of viral variation. under the pressure of herd immunity, the s gene of pedv mutates frequently, with some of the missense mutations altering viral antigenicity to aid in the virus's escape from preexisting immunity. thus, periodic vaccine updates may be required to ensure sufficient efficacy against emerging virus variants [ ] . in this study, our result revealed that there is little variation in the s gene among these pedv strains. moreover, s protein is implicated in virulence, which was found out in the other studies that s protein can be variant readily while receiving immune pressure [ ] . mutations, including deletions and/or insertions, in the s protein may change the pathogenicity and tissue tropism of coronaviruses [ ] . comparing to the p strain, there was no nucleotides insertion and deletion in the s gene of the p strain. however, we found that there were four amino acid mutations (d a, f r, s r and c g) between the p strain and the p strain. fig. phylogenetic tree analysis of the whole genomes of different pedv strains. phylogenetic analysis conducted for the whole-genome nucleotide sequence of the pedv strains reported in our study and the reference pedv strain. using the mega . software, the adjacency method was used to construct the tree. the numbers at the branch are the guided values (%) after copies. the front ends of the three different sub-strains have been labeled. pedv, porcine epidemic diarrhea virus these mutations have not been reported previously. this finding indicates that these mutations in the s gene may be related to viral pathogenicity. the orf protein was reported to function as an ion channel and can prolongs s-phase, facilitates formation of vesicles and thus to regulate virus production [ ] . it has been speculated that the deletion or mutation of a base in the orf region promotes the adaptation of viral cells and changes in viral titers [ ] . in this study, the t m mutation was detected in orf of p . furthermore, mutations of amino acids in the orf ab, m, and e regions of p indicate that multiple mutation combinations in the genome cause complete decay of the pedv strain via various molecular mechanisms [ ] . these mutations found in orf ab, m, orf and e proteins have not been reported previously. further studies using reverse genetics are required to determine whether a particular or a combination of genetic changes (point mutations, deletions, and insertions) in pedv strains alter viral infectivity, pathogenicity, and replication efficiency [ ] . in conclusion, an attenuated strain of pedv with better cell adaptability and higher titers than the virulent strain was obtained via cell passaging. 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vaccines for porcine epidemic diarrhea virus and other swine coronaviruses genetic epidemiology of porcine epidemic diarrhea virus circulating in china in - based on spike gene trypsin-independent porcine epidemic diarrhea virus us strain with altered virus entry mechanism improvements in methods for calculating virus titer estimates from tcid and plaque assays isolation and characterization of a variant porcine epidemic diarrhea virus in china a highly pathogenic strain of porcine deltacoronavirus caused watery diarrhea in newborn piglets oral administration of coated pedv-loaded microspheres elicited pedv-specific immunity in weaned piglets genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype b porcine epidemic diarrhea virus attenuation of an original us porcine epidemic diarrhea virus strain pc a via serial cell culture passage sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis cell attenuated porcine epidemic diarrhea virus strain zhejiang provides effective immune protection attributed to dendritic cell stimulation cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus pedv orf encodes an ion channel protein and regulates virus production preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank wen's group academy, wen's foodstuffs group. authors' contributions fc and yw conceptualized the study. yw and wl devised the experimental methods. yw and zcx curated the data. fc, yw, hqs, qhl, and qfz provided resources. yw prepared the original manuscript draft. fc reviewed the manuscript and edited it. all authors read and approved the final manuscript. the datasets analyzed during this study are available from the corresponding author on reasonable request. the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk- - ). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. the authors declare that they have no competing interests. key: cord- -a ztdpg authors: zhang, yiqiang; liu, yongsheng; liu, wenqian; zhou, jianhua; chen, haotai; wang, yin; ma, lina; ding, yaozhong; zhang, jie title: analysis of synonymous codon usage in hepatitis a virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: a ztdpg background: hepatitis a virus is the causative agent of type a viral hepatitis, which causes occasional acute hepatitis. nevertheless, little information about synonymous codon usage pattern of hav genome in the process of its evolution is available. in this study, the key genetic determinants of codon usage in hav were examined. results: the overall extent of codon usage bias in hav is high in picornaviridae. and the patterns of synonymous codon usage are quite different in hav genomes from different location. the base composition is closely correlated with codon usage bias. furthermore, the most important determinant that results in such a high codon bias in hav is mutation pressure rather than natural selection. conclusions: hav presents a higher codon usage bias than other members of picornaviridae. compositional constraint is a significant element that influences the variation of synonymous codon usage in hav genome. besides, mutation pressure is supposed to be the major factor shaping the hyperendemic codon usage pattern of hav. hepatitis a virus (hav), the causative agent of type a viral hepatitis, is an ancient human virus that was first identified in the stools of infected people in [ ] . hav is a non-enveloped, single-stranded positive-sence rna virus which belongs to order picornavirales, family picornaviridae, the genus hepatovirus in virus taxonomy [ ] [ ] [ ] . the genome of hav is approximately nucleotide in length and contains a large open-reading frame (orf) encoding a polyprotein in which the major capsid proteins represent the amino-terminal third, with the remainder of the polyprotein comprising a series of nonstructural proteins required for hav rna replication: b, c, a, b, c pro and d pol . based on the studies of genetics, hav was proposed to divide into six different genotypes [ ] . however, there is only one known serological group of human hav [ , ] . although hav causes occasional, dramatic disease outbreaks of acute hepatitis with fatal outcomes in otherwise healthy adults as well as isolated severe cases of hepatitis, it has never been associated with chronic liver disease [ ] . as we all know, the genetic code chooses codons to represent standard amino acids and stop signals. these alternative codons for the same amino acid are termed as synonymous codons. synonymous mutations tend to occur in the third base position, but the cases can be interchanged without altering the primary sequence of the polypeptide product. some reports indicate that synonymous codons are not chosen equally both within and between genomes [ ] [ ] [ ] [ ] [ ] . in general, codon usage variation may be the product of natural selection and/or mutation pressure for accurate and efficient translation in various organisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is well known that codon usage variation is considered as an indicator of the forces shaping genome evolution. in addition, compared with natural selection, mutation pressure plays an important role in synonymous codon usage pattern in some rna viruses [ , , ] . nevertheless, little information about codon usage pattern of hav genome including the relative synonymous codon usage (rscu) and codon usage bias (cub) in the process of its evolution is available. in this study, the key genetic determinants of codon usage index in hav were examined. the values of nucleotide contents in complete coding region of all hav genomes were analyzed (table ) . evidently, (c+g)% content fluctuated from . to . , with a mean value of . and s.d of . , indicating that nucleotides a and u were the major elements of hav genome. comparing the values of a %, u %, c % and g %, it is clear that u % was distinctly high, and c % was the lowest of all. the (c +g )% in complete coding region of each hav genome fluctuated from . to . , with a mean value of . and s.d of . . and the effective number of codons (enc) values of these hav genomes fluctuated from . to . , with a mean value of . and s.d. of . . the enc values for these hav genomes were a little low indicating that the there is a particular extent of codon preference in hav genome. the details of the overall relative synonymous codon usage (rscu) values of codons in hav genomes were analyzed (table ) . most preferentially used codons in hav are a-ended or uended codons except the gln and leu whose optimized codons are cag and uug ending by g, respectively. interestingly, hav prefers u-ended optimized codons to a-ended codons. to investigate the major trend in codon usage variation among hav, coa was used for all hav complete coding regions selected for this study. coa detected one major trend in the first axis (ƒ' ) which accounted for . % of the total variation, and another major trend in the second axis (ƒ' ) which accounted for . % of the total variation. a plot of the first and second principal axes of the complete coding region of each gene was shown in figure . it is clear that coordinate of each gene is relatively isolate except the australia isolates, brazil isolate and one russia isolate. nevertheless, these relatively isolated spots tend to cluster into several groups according to the same genotype. but mbb which isolated from north africa had a special codon usage pattern contrasting with the other ib strains. all above imply that these strains of hav isolated from different places, even the same genotype, have different trend in codon usage variation. interestingly, the pattern of codon usage in vaccine strain h change to mbb-like pattern after continuous culturing in a human diploid cell line (kmb ), i.e. h k and h k , suggesting that host was an element that could dramatically influence the codon usage pattern. in order to analyze whether the codon usage variation of hav genome was regulated by natural selection or mutation pressure, the a%, u%, c%, g% and (c+g)% were respectively compared with a %, u %, c %, g % and (c +g )% (table ). there was a complex correlation existing in nucleotide compositions. in detail, a %, c % and g % have a significant negative correlation with c%, u% and a%, respectively. these data suggest that the nucleotide constraint may influence synonymous codon usage. however, a % has non-correlation with u%, and u % has non-correlation with a%, c%, g % and (c+g)%, respectively, which haven't indicated any peculiarity about synonymous codon usage. furthermore, c % and g % have non-correlation with g% and c% respectively, indicating these data probably don't reflect the true feature of synonymous codon usage as well. therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. details of correlation analysis between the first two principle axes (ƒ' and ƒ' ) of each hav genome in coa and nucleotide contents were analyzed (table ). in surprise, only a % has a significant correlation with both principle axes which represent the major trend in codon usage variation, suggesting that nucleotide a is the major factor influencing the synonymous codon usage pattern of hav genome. however, interestingly, although the (ƒ' ) value has non-correlation with base nucleotide c and g contents on the third codon position respectively, it is observably related to (c +g )%, suggesting that codon usage patterns in hav probably be correlated with (c +g )% to a specific extent. overall, compositional constraint is a factor shaping the pattern of synonymous codon usage in hav genome. mutational bias is another main factor leading to codon usage variation enc-plot was considered as a part of the general strategy to investigate patterns of synonymous codon usage. the enc-plots of the genes, whose codon choice is constrained only by a c +g composition, will lie on or just below the curve of the predicted values (wright, ) . enc values of each hav genome were plotted against its corresponding (c +g )%. all of the spots lie below the curve of the predicted values, as shown in figure , suggesting that the codon usage bias in all these hav genomes is principally influenced by the mutational bias. overtime, there have been more and more features that are unique to hav within the family picornaviridae, including its tissue tropism, its virion morphogenesis, its genetic distance from other members of this family, the important details of the processing of the viral polyprotein and the interactions of the virus with host cells [ ] . after we analyzed synonymous codon usage in hav ( [ ] . as a result, hav codon usage has evolved to be complementary to that of human cells, never adopting codons those abundant for the host cell, even in some instances using these abundant codons as rare codons [ ] . since the variation and evolution of virus generally appear in the changes of virus genome composition, compositional constraint was assumed to be closely correlated with the synonymous codon usage pattern [ , , [ ] [ ] [ ] [ ] . nucleotide u content was the highest, and the ratio of u % was much higher than the other base composition on the third codon position (table ) , which interpreted why most of the preferentially used codons are u-ended codons ( table ). despite the ratio of u % was the highest, the major compositional constraint, which shaping the synonymous codon usage pattern of hav genome, was from the percent of nucleotide a on the third codon position (table ) . moreover, two principle axes (ƒ' and ƒ' ) are not correlated with the other base compositions except nucleotide a (table ). this discovery was different from many reports which suggest that c+g compositional constraints were the major factor influencing codon usage bias in virus genome [ , , ] . therefore, we supposed that the compositional constraint was from not only c+g contents but also a and/or u contents. in addition, we found that a % has a remarkable correlation with (c+g)% (table ) . hence, we could infer that a % could influence the synonymous codon usage pattern through coordinating the contents of (c+g)%. moreover, each composition was closely correlated with one of the other compositions, and each composition has a striking negative correlation with the other compositions. the (c +g )% was correlated with all the base compositions especially u and c contents. all these data suggest that there were kinds of complex and fantastic interrelations existing among these base compositions to regulate the codon usage bias. in brief, compositional constraint can indeed determine the variation of synonymous codon usage in virus genome. mutational pressure and natural selection are generally thought to be the main factors that account for codon usage variation between genes in different table summary of correlation analysis between the a, u, c, g contents and a , u , c , g organisms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we wished to determine which should be responsible for the extreme codon usage bias in hav. in the present study, the mutational pressure was determined to be the more important factor for the codon usage bias in hav, which is shown in figure , indicating that the codon usage in hav genome is influenced by the c+g content which is usually assumed to be the result of mutational pressure. actually, it is previously reported that mutation pressure rather than natural selection is the most important determinant of the codon bias in human rna viruses [ ] . since mutation rates in rna viruses are much higher than those in dna viruses [ ] , it is understandable that mutational pressure is the major factor of shaping codon usage pattern in the hav strains included in our study. despite this, hav does not appear to undergo the rapid accumulation of genetic changes seen in many rna viruses. because hav exploits a very low translation rate and a very low replication rate to promote and ensure its survival [ , ] , it shows a quite low mutation rate than other members of the family picornaviridae [ , ] . since hav mutation rate is much lower than other members of the family picornaviridae, how does it form such a higher codon usage bias than other members of the family? furthermore, how does it form kinds of trends in codon usage variation among different stains (shown in figure ) in the condition of the similar nucleotide contents (table )? this could be ascribed to the distinct endemicity of hav, which is speculated from the result of coa. early comparative studies of the nucleotide sequences of different human hav strain suggested that sequence correlation could be correlated with the geographical origin of viruses [ , ] . it is well known that quasispecies dynamics is characterized by continuous generation of variant viral genomes, competition among them, and selection of the fittest mutant distributions in any given environment. as other rna viruses, hav exists in vivo as distributions of closely related variant referred to as quasispecies [ , ] . hav strains maintained their low rate of accumulating mutations over a long period of time so that it developed specific ecological niches [ ] . because of surviving in different geographical area, different human race and different rounds of replication, the extreme codon usage bias of hav was established over a long time. moreover, in the context of a very low mutation rate, the extreme codon usage bias of hav was conserved so that a distinct endemicity was generated. hav presents a higher codon usage bias than other members of picornaviridae. the most important determinant of the high codon bias in hav is mutation pressure which is also the main element shaping the hyperendemic codon usage pattern of hav despite the mutation rate of hav is quite low. besides, compositional constraint is another factor influencing the synonymous codon usage in hav. although basic knowledge of codon usage patterns of hav and the factors regulating the synonymous codon usage are demonstrated in our present study, more comprehensive analysis is necessary for revealing the deeper characteristic of synonymous codon usage in hav genome. the available complete rna sequences of hav were obtained from genbank randomly in october . the serial number (sn), genbank number, genotype and other detail information are listed in table . relative synonymous codon usage values of each codon in a gene were calculated to investigate the characteristics of synonymous codon usage without the confounding influence of amino acid composition of different gene sample [ ] . the rscu value of the ith codon for the jth amino acid was calculated as: where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. when the codon with rscu values close to . , it means that this codon is chosen equally and randomly. the enc was calculated to quantify the codon usage bias of an orf [ ] , which is the best estimator of absolute synonymous codon usage bias [ ] . the larger extent of codon preference in a gene, the smaller the enc value is. and the index gc s was used to calculate the fraction of the nucleotides g+c at the synonymous third codon position (excluding met, trp, and the termination codons). multivariate statistical analysis can be used to explore the relationships between variables and samples. in this study, correspondence analysis was used to investigate the major trend in codon usage variation among genes. in this study, the complete coding region of each gene was represented as a dimensional vector, and each dimension corresponds to the rscu value of one sense codon (excluding met, trp, and the termination codons) [ ] . correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern [ ] . this analysis was implemented based on the spearman's rank correlation analysis way. all statistical processes were carried out by with statistical software spss . for windows. hepatitis a: detection by immune electron microscopy of a virus-like antigen associated with a cute illness classification of hepatitis a virus as enterovirus type and of hepatitis b virus as hepadnavirus type i taxonomic classification of hepatitis a virus classification and nomenclature of viruses: fifth report of the international committee on taxonomy of viruses evidence of recombination in natural populations of hepatitis a virus similarities of two hepatitis a virus strains antigenic relatedness of two strains of hepatitis a virus determined by cross-neutralization hepatitis a virus: from discovery to vaccines codon catalog usage and the genome hypothesis variation in g+c content and codon choice: differences among synonymous codon groups in vertebrate genes evolution of codon usage patterns: the extent and nature of divergence between candida albicans and saccharomyces cerevisiae the relationship between synonymous codon usage and protein structure tissue-specific differences in human transfer rna expression codon usage in regulatory genes in escherichia coli does not reflect selection for 'rare' codon codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes what drives codon choices in human genes? ribosome traffic in e.coli and regulation of gene expression analysis of synonymous codon usage in sars coronavirus and other viruses in the nidovirales analysis of synonymous codon usage in h n virus and other influenza a viruses synonymous codon usage in environmental chlamydia uwe reflects an evolution divergence from pathogenic chlamydiae mutation pressure shapes codon usage in the gc-rich genome of foot-and-mouth disease virus codon usage in nucleopolyhedroviruses the extent of codon usage bias in human rna viruses and its evolutionary origin genetic variability and molecular evolution of hepatitis a virus genome variability and capsid structural constraints of hepatitis a virus codon usage and replicative strategies of hepatitis a virus codon usage and genome composition compositional constraints and genome evolution synonymous codon usage in adenoviruses: influence of mutation, selection and protein hydropathy analysis of synonymous codon usage in human bocavirus isolates mutation rates among rna viruses analysis of sequential hepatitis a virus strains reveals coexistence of distinct viral subpopulations genetic relatedness of hepatitis a virus strains recovered from different geographical regions molecular epidemiology of human hepatitis a virus defined by an antigen-capture/polymerase chain reaction method epidemiologic patterns of wild-type hepatitis a virus determined by genetic variation the 'effective number of codons' used in a gene an evaluation of measures of synonymous codon usage bias multivariate analysis newyork analysis of synonymous codon usage in hepatitis a virus this work was supported in parts by grants from national key technologies authors' contributions yqz conceived of the study, downloaded these sequences, calculated the data, analyzed the results and drafted the manuscript; ysl conceived of the study, supervised the research, analyzed the results and helped draft the manuscript; jhz calculated and visualized the data; wql, htc, yw, lnm and yzd assisted with data analysis; jz supervised the research and helped draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - in m w authors: abdel-moneim, ahmed s; el-kady, magdy f; ladman, brian s; gelb, jack title: s gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: in m w background: infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. ibv is endemic in probably all countries that raise chickens. it exists as dozens of serotypes/genotypes. only a few amino acid differences in the s protein of vaccine and challenge strains of ibv may result in poor protection. tropism of ibv includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. results: infectious bronchitis virus (ibv) strain closely related to massachusetts (mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. the isolate was serologically identified by dot-elisa and further characterized by rt-pcr then genotyped using s gene sequence analysis. alignment of the s sequence of the isolate with ibv strains revealed high homology to isolates related to mass serotype. inoculation with the strain reproduced the disease in experimental -day-old chickens and resulted in % mortality, severe renal and moderate respiratory distresses. marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. a protection study using the h live attenuated vaccine showed low protection rate in spite of high s sequence homology ( %). protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as ibv antigens detection by immunofluorescent antibody technique in kidney sections. conclusion: periodical evaluation of cross-protective capabilities of ibv vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of ibv. avian infectious bronchitis virus (ibv) is a highly contagious pathogen of chickens that replicates primarily in the respiratory tract and also in some epithelial cells of the gut, kidney and oviduct [ ] . ibv is a virus member of genus coronavirus, family coronaviridae, order nidovirales [ ] . the virus possesses a positive stranded rna genome that encodes phosphorylated nucleocapsid pro-tein (n), membrane glycoprotein (m), spike glycoprotein (s) and small membrane protein (e). the spike glycoprotein is post-translationally cleaved into two subunits, s and s [ , ] . the s protein forms the n-terminal portion of the peplomer and contains antigenic epitopes mainly within three hvrs [ ] [ ] [ ] . neutralizing and serotype specific epitopes are associated within the defined hvrs [ , , ] . variation in s sequences [ ] [ ] [ ] , has been recently used for distinguishing between different ibv serotypes. diversity in s probably results from mutation, recombination and strong positive selection in vivo [ ] . antigenically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. the generation of genetic variants is thought to be resulted from few amino acid changes in the spike (s) glycoprotein of ibv [ , ] . in egypt, isolates related to massachusetts, d , d , d- , / and the novel genotype; egypt/beni-suef/ were isolated from different poultry farms [ ] [ ] [ ] [ ] . the commonly used ibv attenuated vaccine is h while the mass (m ) strain is commonly used in inactivated vaccines. in the present study, egypt/f/ was isolated from day-old broiler chickens in fayoum governorate, identified by dot-elisa, rt-pcr and sequenced to determine its serotype. pathogenicity test to -day-old chickens and protection afforded by the commonly used h live attenuated vaccine were also performed. the allantoic fluid of the first chicken embryo passage of egypt/f/ was harvested at h pi. four additional egg passages were performed. five eggs of the th passage were incubated till being -day-old and all of them ( %) showed typical lesions of the ibv (stunting and dwarfing). the virus identity was ascertained by performing dot-elisa on the cam homogenate (fig. ) . polymerase chain reaction and s gene cycle sequencing rt-pcr of egypt/f/ resulted in a product of base pairs using s primers oligo ' and oligo '. egypt/f/ is closely related to the beaudette us reference strain; % nucleotide identity and % amino acid identity (table and fig. ). it showed % similarities both in nucleotides and amino acids to h and % nucleotide and % amino acid homology to m (table ) . egypt/ f/ showed point mutations from h ; silent and non silent mutations. on the other hand, it showed point mutations; silent and non silent mutations from m (fig. , ) . sixteen potential glycosylation sites were found in egypt/f/ while were found in h and m (fig. ) . all potential glycosylation sites found in egypt/f/ were shared with those found in h and m (fig. ) . chickens inoculated with egypt/f/ exhibited snicking and rales in approximately % of infected birds at rd day of inoculation. conjunctivitis was observed in / at rd day pi that was elevated to / by the th day pi of infected birds whereas no birds exhibited watery eyes. six birds were dead after egypt/f/ experimental infection; birds in the th day pi and birds in the th day pi. postmortem examination of dead birds, revealed petechial haemorrhages in larynx and thymus, severe congestion of liver, spleen and lungs as well as renal haemorrhages. these changes appeared but in milder form in birds sacrificed at days pi. histopathological examination of sacrificed birds at days pi and freshly dead birds at days pi revealed mucus, marked loss of cilia, desquamation, mononuclear infiltration, epithelial hyperplasia and vascular congestion of the trachea (fig. ). kidneys showed severe changes including haemorrhages, degenerative changes in renal tubules and hypercellularity of the renal glomeruli as well as focal lymphocytic infiltration (fig. ) . in general the tracheal and renal histopathological lesions were more severe in dead birds (fig. b , d) than birds sacrificed at days pi (fig. a, c) . chickens vaccinated with h (group a) showed . % protection ( / ) by virus reisolation procedure and . % ( / ) protection by histopathology after challenge with egypt\f/ while all control unvaccinated birds (group b) were not protected ( / ) ( table ) . on day four pi, the kidneys of ( / ) birds in group a (vaccinated and challenged with ibv) showed focal lymphocytic infiltration, urates deposition and degenerative changes in renal tubules while birds of group b (unvaccinated and challenged with ibv) showed multifocal lymphocytic infiltration, urates deposition and degenerative changes in renal tubules in / birds, while only focal lymphocytic infiltration, urates deposition and degenera-dot-elisa shows positive reaction in tested (chorioallantoic membrane homogenate) and control positive sample control +v e tested sample control -ve tive changes in renal tubules were observed in / birds. birds in group c (vaccinated unchallenged group) did not show any abnormalities ( / ) either in tracheae or kidneys. immunofluorescenct assay on kidneys of challenged and unchallenged groups showed that all challenged unvaccinated and / of challenged vaccinated groups possessed kidney immunofluorescence. none of control unchallenged groups possessed kidney immunofluorescence (table ). in this study, an egyptian ibv strain; egypt\f/ was isolated from a tissue pool of kidney and trachea from unvaccinated broiler flock with a history of respiratory and renal disease. the strain produced typical lesions of ibv in inoculated embryos and identified as ibv by dot-elisa and rt-pcr. the isolate was found to be devoid of major concomitant viruses; avian influenza virus, newcastle disease virus, infectious laryngotracheitis virus, reovirus and adenovirus (data not shown). s sequence analysis of egypt/f/ revealed its close relatedness to mass serotype. it showed high nucleotide similarities to gx - .china ( % nucleotide and amino acid identities), beaudette-us ( % nucleotide and % amino acid identities), is/ / ( % nucleotide and % amino acid identities), h ( % nucleotide and amino acid identities) and m ( % nucleotide and % amino acid identities). ibv s gene sequence relationships expressed as a phyloge-netic tree of egypt/f/ isolate and selected ibv reference strains figure ibv s gene sequence relationships expressed as a phylogenetic tree of egypt/f/ isolate and selected ibv reference strains. it is known that the most severe clinical response of ibv appears in very young chickens and severity is alleviated in older chickens [ , ] . this fact explains the high mortality rate observed in -day-old chickens that experimentally inoculated with egypt/f/ compared to mortality pattern in the original flock ( -day-old chickens). the presence of acute interstitial nephritis on days and post infection indicated that egypt\f is a nephrogenic ibv. the microscopic findings of the renal tubules matched the general findings recorded with nephrogenic ibv strains [ , ] . the microscopic findings in tracheal sections appeared similar to those recorded by [ , ] including: loss of cilia, degenerative changes of the tracheal mucosa, irregular loss of epithelium, desquamation of the sloughed epithelium in the tracheal lumen and lymphocytic infiltration that ranged from focal aggregation to diffuse massive infiltration. severe renal haemorrhages observed grossly and in hisopathological sections of birds dead after experimental infection with egypt/f/ denote that deaths resulted from acute renal failure. our finding regarding the presence of petechial haemorrhages in larynx and thymus as well as severe congestion of liver, spleen and lungs in birds dead after ibv experimental infection is in agreement with [ , ] who confirmed the presence of ibv viral antigens in such organs. evaluation of the immune response to ibv vaccination is based on several criteria including: clinical signs, tracheal nucleotides identities of egypt/f/ with commonly used vaccine strains sequences figure nucleotides identities of egypt/f/ with commonly used vaccine strains sequences. dots indicate residues identical to egypt/f/ . bold letters denotes codon areas. shaded letters denote sites of differences. histological lesion, virus neutralization, virus re-isolation from trachea, antigen detection in trachea and/or kidney by immunofluorescent or immunoperoxidase techniques [ ] [ ] [ ] [ ] [ ] . in this study we used tracheal histological lesion and virus re-isolation as parameters for tracheal protection. kidney histopathology and antigen detection by ifa were used as indicators of kidney protection. complete protection is expected upon using closely related vaccine strain as the degree of cross protection among ibv strains generally reflects the similarities between the s proteins [ , ] . the re-isolation of egypt/f/ from the trachea of vaccinated birds and the presence of tracheal and renal microscopic lesions as well as viral antigen in kidneys (by ifa) in h vaccinated birds denote lack of complete protection afforded by h vaccination. h is a mild vaccine and it is possible that the challenge virus was too virulent for the level of immunity that the vaccine produced in these young chickens. other possible consideration includes that baby chickens are not fully immunocompetent at one-day of age, the time that they were vaccinated for the protection study experiment. however, commercial broiler chickens possess maternally derived antibodies, are routinely vaccinated at one-day of age [ , ] without apparent interference by the maternal derived antibodies in the development of active immunity, at least in the respiratory tract that measured by challenge [ ] . on the other hand, variable results were recorded regarding homologous protection of ibv. cavanagh et al. [ ] inoculated groups of chickens with the virulent uk/ / isolate and challenged with isolates that differed by up to % of s amino acids. challenge with two variants ( % s identity with uk/ / ) resulted in challenge scores virtually the same as with the homologous challenge however, challenge with two others iso-lates ( % and % s identity, respectively), resulted in less cross-protection, although the numbers were not statistically significantly different. in the s subunit, three hvrs are located within amino acids - , - and - [ ] [ ] [ ] . hvr and hvr contain sequences that have been associated with specific ibv serotypes [ , ] as well as serotype specific neutralizing epitopes [ , , ] . ibv serotypes commonly differ by to % in s [ , ] but some serotypes differ in s by as little as % [ ] . although h showed % amino acid and nucleotide identity to egypt/f/ , it possesses different nucleotides that resulted in amino acid substitutions. among such amino acids, one is located in hvr , four in hvr and one in hvr (fig. ) . the region between amino acid residues - has been previously identified as a possible region involved in the differing pathogenicity of gray and non-virulent jmk strains [ ] . egypt/f/ possesses different amino acid; phenylalanine within this region at positions and instead of serine and leucine in h respectively. it is apt to mention that some serotypes differ in s by as little as amino acids [ ] , suggesting that only a few epitopes may induce most of the vn antibody [ ] . egypt/f/ is a nephropathogenic ibv strain closely related to mass serotype. vaccination by h did not provide satisfactory protection against challenge with egypt/ protective capabilities of such vaccine(s) versus recently recovered field isolates in order to ensure optimum control of ibv. embryonated chicken eggs spf ece obtained from nile spf (koom oshiem, fayoum, egypt) were used for isolation of the field isolate, serial passages, titration of the seed stocks of egypt/f/ and vaccine strain (h ), as well as virus re-isolation attempts following challenge in the protection study. sixty nine commercial -day-old chickens (el-waddi co, egypt) were reared under strict hygienic conditions in sep-arate rooms and used in both virulence test and protection study. rabbit anti-ibv polyclonal antiserum raised against vero adapted h vaccine was prepared previously in our lab [ ] and used for detection of ibv antigens in both dot-elisa and indirect immunofluorescent antibody technique. infectious bronchitis was diagnosed during augest in fayoum governorate, egypt. the outbreak occurred in -day-old commercial broiler farm with no previous ibv vaccination. the flock was vaccinated against newcastle disease and infectious bursal disease viruses at and days of age respectively. the total flock density was birds. the first signs were depression and respiratory distresses including sneezing, coughing and rales. other signs included conjunctivitis and watery eyes. within a period of days after the appearance of the disease, the mortality rate increased to % of the flock density. postmortem examination of dead birds revealed increased tracheal mucus, severe renal congestion, urates filled ureters as well as congestion in liver and spleen. egypt/f/ was isolated from -day-old broiler chickens suffering from both respiratory and renal distresses from fayoum governorate in . a kidney homogenate ( % in sterile pbs) and a tracheal scraping suspension were pooled, centrifuged at × g for min. the supernatant fluid was inoculated into chorioallantoic sac of day-old spf ece. allantoic fluid was harvested after h and was used for re-passage into ece. five eggs of the th egg passage were incubated till being -day-old and examined for typical lesions of ibv (stunting, curling and urates deposition in ureters). a dot-elisa was performed according to [ ] . briefly, ncm of convenient size was cut, marked with waterproof ink for identification and then soaked for min. in distilled water. ncm was laid on absorbent paper and airdried for min. approximately μl of the extracted viral rna was used to synthesize cdna. amplification of the s gene was performed using s oligo ' ( '-cataactaacataag-ggcaa- ') and s oligo ' ( '-tgaaactgaacaaaagac- ') primers [ , ] . pcr of s gene was performed as described [ ] with the exception that extension was performed at °c. s pcr product was cut from . % agarose gels, purified with the qiaquick gel extraction kit (qiagen, inc.) and the dna was quantitated as described [ ] . purified rt-pcr product was sequenced in the forward and reverse directions using the same primers. sequencing was performed as described [ ] . a blast ® analysis [ ] was initially performed using the s sequence of egypt/f/ (dq ) to establish its identity to genbank accessions. a comparative analysis of s sequences was performed using the clustal w multiple sequence alignment program, version . [ ] . the tree was constructed using the neighbour-joining program [ ] . ibv s sequences representative to genotypes used for the alignments were obtained from the genbank and embl database. forty -day-old chickens were used. thirty chickens were infected by intraocular instillation of eid / μl of infectious bronchitis virus taxonomy coronaviruses in poultry and other birds amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions identification of amino acids involved in a serotype and neutralization specific epitope within the s subunit of avian infectious bronchitis virus analysis of the serotypespecific epitopes of avian infectious bronchitis virus strains ark and mass epitopes on the peplomer protein of infectious bronchitis coronavirus strain m as defined by monoclonal antibodies a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism differentiation of infectious bronchitis virus serotypes using the polymerase chain reaction and restriction fragment length polymorphism analysis identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s- gene relationship between sequence variation in the s spike protein of infectious bronchitis virus and the extent of cross-protection in vivo location of the amino-acid differences in the s spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide present status of infectious bronchitis in egypt studies on the epidemiology and means of control ofinfectious bronchitis disease in chickens in egypt isolation and identification of egypt/beni-suef/ a novel genotype of infectious bronchitis virus urolithiathis in white commercial egg laying chickens associated with an infectious bronchitis virus differences inpathogenicity for young chickens among field isolates of infectious bronchitis virus studies on the prevalence and epidemiology of infectious bronchitis in broiler chickens in el-minia governorate the histopathology of infectious bronchitis in fowls infected with a nephrotropic t strain of virus comparison of the nephropathogenicity of four strains of infectious bronchitis virus the control of avian infectious bronchitis/nephrosis in australia in vitro characterization and pathogenesis of egypt/beni-suef/ ; a novel genotype of infectious bronchitis virus criteria for examining the immune response to infectious bronchitis virus some characteristics of isolates of infectious bronchitis virus from commercial vaccines the immune response to infectious bronchitis virus determined by respiratory signs, virus infection, and histopathological lesions challenge experiments to evaluate cross-protection induced at the trachea and kidney level by vaccine strains and belgian nephropathogenic isolates of avian infectious bronchitis virus protection of chickens following live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/ severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus vaccination of day-old broilers against infectious bronchitis: effect of vaccine strain and route of administration vaccination of one day-old broilersagainst infectious bronchitis by eye drop application or coarse droplet spray and the effect of revaccination by spray lack of correlation between infectivity, serological response, and challenge results in immunization with an avian infectious bronchitis vaccine comparison of the spike precursor sequences of coronavirus ibv strains m and / with that of ibv beaudette phylogeny of antigenic variants of avian coronavirus ibv. virology serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s- ) gene molecular cloning and sequence comparisonof the s glycoprotein of the gray and jmk strains of avian infectious bronchitis virus recombinant infectious bronchitis coronavirus beaudette with the spike protein gene of the pathogenic m strain remains attenuated but induces protective immunity a dot-immunobinding assay for monoclonal and other antibodies basic local alignment search tool bcm search launcher: multiple sequence alignments serum antibody responses of chickens following sequential inoculation with different infectious bronchitis virus serotypes evaluation of ciliary movement in tracheal rings to assess immunity against infectious bronchitis virus diagnosticmethods in clinical virology egypt/f/ according to [ ] while other birds were kept as control uninfected group. clinical signs and gross postmortem lesions as well as mortalities were recorded. microscopic examinations of both tracheae and kidneys were performed at and days post infection. twenty nine commercial -day-old chickens were used to evaluate the protection provided by h vaccination against challenge with egypt/f/ . birds were divided into three groups; a (n = ), b (n = ), c (n = ). vaccination was performed at day by eye drop application. single dose of h vaccine (nobilis, intervet, the netherlands bv) was used for each bird in groups a and c according to manufacturer's instructions while birds in group b were kept as unvaccinated control. four weeks post vaccination, chickens in group a and b were challenged by eye drop with egypt/f/ ( eid per bird) while birds in group c were not challenged and kept as vaccinated unchallenged control. tracheae of all birds from all groups were collected four days post challenge for virus reisolation attempts and histopathological examination. tracheal scrapings were emulsified in ml of sterile pbs and centrifuged at × g for min. virus reisolation attempts were performed by inoculating - , -day-old spf ece by the supernatant fluid of each sample as described [ ] . embryos were examined for typical lesions of ibv. for histopathological examination, tracheae were fixed in formalin, processed routinely for histopathology and stained with haematoxylin and eosin. the trachea from each bird was examined microscopically and assigned lesion scores of - with = none, = focal, = multifocal, = diffuse. tracheae were scored for the amount of mucous, loss of cilia, epithelial hyperplasia, necrosis, lymphocyte and heterophil infiltrations as well as the extent of tissue reaction. the scores for each bird were added and the mean score for the birds in each group was calculated [ ] . kidney samples were also taken days post challenge and examined microscopically for tubular degeneration and inflammation consistent with interstitial nephritis. focal, multifocal and diffuse were used to assign kidney histopathology. the presence of viral antigens in kidneys was screened by immunofluorescent antibody technique. it was performed according to [ ] to detect viral antigens in the kidneys of birds after challenge with egypt/f/ in protection study. briefly, deparaffinized slides were incubated with rabbit anti-ibv antibodies ( : ) for h and subsequently with fitc-conjugated goat anti-rabbit antibody (kirkegaard and perry laboratories, gaithersburg, md.) ( : ) for h. both primary and secondary antibodies were diluted in pbs. slides were rinsed three times ( min./single wash) with pbs after each step. slides were then mounted using glycerol/pbs (without allowing the slides to dry) and examined under fluorescent microscopy.abbreviations cam, chorioallantoic membrane; ece, embryonated chicken eggs; eid, egg infective dose fifty; fitc, fluorescien isothiocynate; hvr, hypervariable region; ibv, infectious bronchitis virus; ncm, nitrocellulose membrane; pbs, phosphate buffer saline; rt, reverse transcriptase; spf, specific-pathogen-free. the author(s) declare that they have no competing interests. asa isolated and serologically characterized egypt/f/ virus and performed virulence test as well as protection study. he also performed multisequence alignment, phylogenetic analysis and drafted the manuscript, mfe provided sample for isolation, helped in performing virulence test and protection study and reviewed the manuscript, jgjr helped in performing rt-pcr, s gene sequence of egypt/f/ and critically reviewed the manuscript, bsl made rt-pcr and s gene sequence of egypt/ f/ . key: cord- -u r usfs authors: yao, li-hong; wang, chao; wei, tian-li; wang, hao; ma, fen-lian; zheng, li-shu title: human adenovirus among hospitalized children with respiratory tract infections in beijing, china, – date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: u r usfs background: human adenoviruses (hadvs) cause a wide range of diseases. however, the genotype diversity and epidemiological information relating to hadvs among hospitalized children with respiratory tract infections (rtis) is limited. here, we describe the epidemiology and genotype distribution of hadvs associated with rtis in beijing, china. methods: nasopharyngeal aspirates (npa) were collected from hospitalized children with rtis from april to march . hadvs were detected by a taqman-based quantitative real-time polymerase chain reaction (qpcr) assay, and the hexon gene was used for phylogenetic analysis. epidemiological data were analyzed using statistical product and service solutions (spss) . software. results: hadv was detected in ( . %) of the npa specimens, with most ( . %, / ) hadv-positives cases detected among children < years of age. hadv-b ( . %, / ) and hadv-c ( . %, / ) were the most frequent. of the hadv-infected cases, ( . %) were co-infected with other respiratory viruses, most commonly parainfluenza virus ( . %, / ) and rhinovirus ( . %, / ). the log number of viral load ranged from . to . copies per ml of npa, with no significant difference between the hadv mono- and co-infection groups. the main clinical symptoms in the hadv-infected patients were fever and cough, and ( . %, / ) were diagnosed with pneumonia. additionally, hadvs were detected throughout the year with a higher prevalence in summer. conclusions: hadv prevalence is related to age and season. hadv-b and hadv-c circulated simultaneously among the hospitalized children with rtis in beijing, and hadv-b type and hadv-c type were the most frequent. first discovered in by rowe et al., human adenoviruses (hadvs) are non-enveloped, double-stranded dna viruses belonging to the mastadenovirus genus (adenoviridae family). they are common pathogens in children and cause a variety of diseases [ ] . hadv accounts for at least to % of pediatric and to % of adult respiratory tract infections (rtis) [ ] . there are currently seven different hadv species (a to g), some of which specifically attack the conjunctiva (species d), the upper and lower respiratory tracts (species b, c and e), and the gastrointestinal tract (species f and g) [ ] . of the adv serotypes, are known to cause human diseases, with adv , , and being the most common types to cause respiratory disease outbreaks [ ] . hadv has caused respiratory tract adenovirus outbreaks in jiangsu and taiwan provinces of china, as well as in korea, singapore and malaysia [ ] [ ] [ ] . such infections have been estimated to cause - % of rtis overall and - % of all pneumonias in city of bethlehem [ ] . although hadvs are associated with mild to moderate disease in most cases, life threatening disease can occur in some patients, particularly if they are immunocompromised [ ] . at present, china has not yet established a nationwide epidemiological surveillance program for adenovirus infections, and infections with it do not need to be legally reported, so the institutions for disease control and prevention cannot conduct early detection screening or issue early warnings. neither are there any u.s. food and drug administration approved antivirals for adenoviral infections [ ] . hadvs play an important role in respiratory infections, particularly in children. therefore, the aim of this study was to evaluate the epidemiological, clinical, and molecular characteristics of hadv infections occurring among children with rtis in a chinese tertiary hospital from april to march . collectively, the findings from this study underscore the importance of monitoring the epidemiology of hadv infections and protecting vulnerable patients as part of the suite of infection prevention strategies in hospitals. the nasopharyngeal aspirate (npa) samples ( ) used in this study were collected from hospitalized children (< years) with rtis at beijing friendship hospital between april and march . informed consent from the parents or guardians of the children enrolled in the study was received. rtis was defined as an illness that presented with at least two of the following clinical presentations: fever, cough, nasal obstruction, expectoration, sneeze and dyspnoea during the previous week. patients, who were diagnosed with pneumonia by chest radiography, were also included in the study, even if they did not show the clinical features described above [ ] . all the specimens were stored at − °c until further processing. demographic and clinical data were obtained from the hospital's database. total viral nucleic acids were extracted from μl of each clinical npa specimen using the qiaamp minelute kit (qiagen, germany) according to the manufacturer's instructions and eluted with μl of nuclease-free water. hadv detection was performed using qpcr assay targeting the highly conserved gene region ( -bp) of the hadv hexon. taqman universal pcr master mix (applied biosystems, usa) was used to amplify hadv hexon dna using specific primers (forward: ′-gccccagtgg tcttacatgcacatc- ′; reverse: ′-gccacggtg gggtttct aaactt- ′) and probe ( ′-fam-tgca ccagacccgggctcaggtactccga- ′-tamra) [ ] . each μl reaction mixture comprised μl of × taqman gene expression master mix, . μl of each primer ( pm), . μl of probe ( pm), . μl of sterile water, and . μl of the nucleic acid components extracted from each sample. the qpcr cycling program was as follows: °c for min, °c for min, followed by cycles of °c for s, and °c for min. samples with a cycle threshold (ct) < were regarded as positive. -fold serial dilutions of pmd -t/hexon plasmid from to copies per μl were prepared to establish a standard curve to measure the hadv load. qpcr were performed using the mx p qpcr system (agilent stratagene, usa). hadv-positive samples were subsequently screened for the following pathogens: influenza a (flua) and b (flub) viruses, parainfluenza viruses (pivs) - , human metapneumovirus (hmpv), human rhinovirus (hrv), wu polyomavirus (wupyv), respiratory syncytial virus (rsv) and human coronaviruses (hcov) nl , oc , e, hku and human bocavirus (hbov), as described previously [ ] [ ] [ ] . additionally, mycoplasma pneumonia is determined by the detection of mycoplasma pneumoniae igm antibody. as for bacterial pneumonia, it was confirmed by sputum culture. nested pcr targeting the hadv hexon gene's hypervariable region was employed for genotyping. the outer primers used were forward ′-gccaccttcttcccc atggc. − ′ and reverse ′-gtagcgttgccggccgagaa- ′, and the internal primers were forward ′-ttcc ccatggcccacaacac- ′ and reverse ′-gcctcgat gacgcc. gcggtg- ′. nested pcr was conducted in μl volume comprising . μl of × ex taq buffer, . μl ( pm) of each primer, . μl of dntp mix, . μl of ex taq dna polymerase, . μl of viral nucleic acid extract or first nested-pcr product, and μl of double-distilled water. the mixtures were amplified with an initial denaturation at °c for min, followed by cycles at °c for min, °c for min, and °c for min, and a min extension at °c. pcr products were analyzed on . % agarose gels, purified, and then confirmed as authentic by sequencing. samples that failed to amplify are defined herein as untyped. all the hexon gene sequences obtained by nested pcr together with the hadv strain sequences available in genbank were used for the phylogenetic analyses. the phylogenetic tree was constructed using molecular evolutionary genetics analysis (mega) . and evaluated using bootstrap replicates to verify the hadv genotypes. positive pcr products were named according to the corresponding serial numbers of the specimens. the hadv detection rates for the different populations and different seasons were compared by a χ test, and the relationship between vomiting and hadv types was statistically analyzed by fisher's exact probability, with statistical significance set at p < . . vector nti . software was used for sequence alignment and mega . software for phylogenetic analysis. epidemiological data were analyzed using statistical product and service solutions (spss) . software. in total, npa specimens were obtained from children with rtis at beijing friendship hospital, among which ( . %) were male and ( . %) were female (table ) , all children survived, and the age range was from day to years of age with a median age of years. from them, ( . %) patients were younger than years old. hadvs were detected in . % ( / ) of the hospitalized children. as shown in table , among the hadvpositive patients, ( . %) were male and ( . %) were female (a male/female ratio of . : ). no significant difference was observed in males and females in the hadv-positive cases (p = . ). hadv was detected among hospitalized children (< years) with rtis at beijing friendship hospital between april and march , and there were significant differences in hadv detection rates among different age groups (p = . ). children under years old accounted for . % ( / ) of the infections. the hadv detection rate peaked in the > to≤ years age group ( . %, / ), while those in the ≤ years old group had a relatively low detection rate of . % ( / ). hadv was detected in every month throughout the study period from april to march , peaking in summer. the hadvs detection frequencies were . % ( / ), . % ( / ), . % ( / ) and . % ( / ) (χ = . , p = . ) in the spring, summer, autumn and winter months, respectively. additionally, the hadv detection rate peaked at . % ( / ) in august (fig. ) . of the hadv-infected cases, ( . %) comprised co-infections with other respiratory viruses including ( . %, / ) with one other virus, ( . %, / ) with two other viruses, ( . %, / ) with three other viruses and ( . %, / ) with four other viruses. the most frequently identified mixed infection was piv ( . %, / ) and hrv ( . %, / ), as shown in table . among the hadv-positive specimens, the log number of hadv genome copies ranged from . to . copies per ml of npa, as determined by the qpcr assay measurements (fig. ) . the log number for the hadv genome copies was . ± . copies/ml of npa in the children infected with hadv only, which is slightly higher than the . ± . copies/ml of npa from those co-infected with hadv and other respiratory viruses; there was, however, no significant difference in the viral loads between the hadv mono-and coinfections (p = . ). sixty-two of the patients ( . %, / ) from the hadv-positive cases were diagnosed with pneumonia, including with bronchopneumonia, with asthmatic bronchitis, with refractory bronchopneumonia, with pneumonia ( with mycoplasma pneumonia, with bacterial pneumonia and with viral pneumonia) and with refractory pneumonia. viral pneumonia and bacterial pneumonia accounts for . % ( / ) and . % ( / ) of the pneumonia, respectively. in addition, the remaining cases were diagnosed with acute tonsillitis ( ), acute upper respiratory infection ( ), acute bronchitis ( ), as well as basing on the -bp fragment of the hexon gene amplified by nested-pcr, hadv-positive samples were sequenced and successfully genotyped. the phylogenetic analyses indicated that cases belonged to species b (hadv-b , hadv-b , and hadv-b ) and belonged to species c (hadv-c , hadv-c , and hadv-c ) ( table ) . six of the samples failed to be typed. the above-mentioned results indicate that species b and c (at least hadv genotypes) circulated simultaneously in beijing, and that hadv-b was the most prevalent genotype, followed by hadv-c (fig. ). the present study was carried out between april and march among hospitalized children with rtis in beijing, china. herein, we describe (i) the prevalence of hadvs in hospitalized children with rtis presenting at beijing friendship hospital during a one-year period; (ii) the clinical spectrum of the hadv-positive rti patients; (iii) the viral co-pathogens present in the hadv infections; and (iv) the genetic diversity of the hadvs. the clinical characteristics of the rtis caused by hadv are very similar to those of influenza, piv and other respiratory tract pathogens, making it difficult to clinically diagnose this type of infection. therefore, effective diagnostic methods are needed for rapid identification and genotyping of hadv. the qpcr assay used herein was established to detect and quantify hadv. a total of npa specimens were screened for the presence of hadv and specimens ( . %, / ) were confirmed to be positive for hadv, which is consistent with prior reports ( . - . %) [ ] [ ] [ ] . the detection rate for hadv varies from region to region in china, and the rate for hospitalized children with acute lower rtis in zhejiang province from to was . , and . % in shenzhen city from to [ , ] . however, it is worth mentioning that such discrepant hadv detection rates can be caused by methodological differences, the number of patients tested, the periods during which the samples are collected, and even a study's duration. previous studies have shown that hadv detection rates are positively correlated with the monthly mean temperature and sunshine duration, and negatively correlated with wind speed; in fact, the higher the air temperature, the higher the detection rate [ ] . our study also revealed that hadv infections occurred throughout the year with the highest prevalence in the summer ( . %, jun to aug), peaking in august ( . %), which is similar to what has been found in tianjin, a northern chinese city, where hadv infections are concentrated during the summer [ ] . however, this finding is discordant with other studies that have reported seasonal peaks for hadv infections in spring in northern china and mexico [ , ] . we found that the hadv infections occurred predominantly in children under years of age ( . %), demonstrating that hadv is an important pediatric pathogen. previous studies drew identical conclusions that most children become infected by hadv at an early age [ , , ] . hadvs can be easily transmitted through fomites contaminated with infectious body fluids. in our study, the hadv detection rate ( . %) was lowest among children under year of age, and the reasons need to be further explored. many studies have reported that the most common hadv species causing rtis in children are b (b , b , b ), c (c , c , c , c ) and e (e ) worldwide [ , ] . hadv , and are the most prevalent species and are associated with severe pneumonia in china [ , ] . in the present study, a total of samples were identified and phylogenetically analyzed based on the hexon gene sequence, which revealed six hadv genotypes and showed that hadv species b and c were the most prevalent, accounting for . and . % of isolates, respectively. similarly to what has been found in asia by other authors, hadv-b was the most common type ( . %, / ) followed by hadv-c ( . %, / ) and c ( . %, / ). hadv-b has been identified as the causative pathogen for severe acute respiratory illness outbreaks in korea [ ] , brazil [ ] and taiwan [ ] , and it was the main type of respiratory hadv infections from to . moreover, hadv-b was the pathogen causing epidemic respiratory disease outbreaks in europe, america, and oceania. last, hadv-b is known to cause a characteristic syndrome in older children and adults, as manifested by acute pharyngo-conjunctival fever, especially after contact with summer camps and swimming pools [ ] . hadv-c and hadv-c have been frequently reported to cause endemic, sporadic and epidemics cases [ , ] . in the present study, the most common diagnosis ( . %) in the hadv-positive cases was pneumonia, with the common signs and symptoms of fever and cough, which is consistent with previous reports [ ] [ ] [ ] . hadv infection is often accompanied by other virus infections. our study showed that piv ( . %, / ) was the major co-infecting pathogen identified followed by hrv ( . %, / ). although the viral load from children mono-infected with hadv was slightly higher than those co-infected with hadv and other respiratory viruses, no significant difference was seen between the two groups. the severity of hadv infection varies according to age, socioeconomic status, environmental status and, above all, the immunological characteristics of the patient. therefore, the etiological significance of coinfections with hadv and other respiratory viruses and its association with disease severity require further study. it was reported hadv can cause more severe illness in immunocompromised patients, so it would be very valuable to know about pre-existing conditions in hadvpositive children. except for cases of asthma, there were no 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clinical features, and prognosis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the department of pediatrics, beijing friendship hospital (capital medical university) for providing the npa specimens. we would also like to thank sandra cheesman, phd, from edanz group (www.edanzediting. com/ac), for editing the english text of this manuscript. authors' contributions ly, fm and cw performed the experiments. fm and lz conceived and designed the experiments. fm, tw, hw and lz analyzed the data. cw, hw and tw contributed reagents/materials/analysis tools. fm and lz wrote the manuscript. all authors read and approved the final manuscript. this work was supported by the key technologies r&d program of the national ministry of science, china ( zx ). the datasets supporting the conclusions of this article are included within the article. the project was approved by the ethical committee of national institute for viral disease control and prevention, china cdc, and the committee's reference number is , ivdc . written informed consent for specimen collection, testing and publication was obtained from the patients' parents or guardians. the authors declare that they have no competing interests. key: cord- -eqqvwwh authors: wang, huanan; cong, feng; guan, jianchi; xiao, li; zhu, yujun; lian, yuexiao; huang, ren; chen, meili; guo, pengju title: development of a sensitive and specific xmap assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: eqqvwwh background: a serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (iltv) and infectious bronchitis virus (ibv) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. method: the microspheres coated with purified recombinant glycoprotein d (gd) of iltv or nucleocapsid (n) protein of ibv were incubated with serum samples. the simultaneous quantification of iltv and ibv antibodies were achieved through the interrogation of microspheres by luminex detection system. . results: this xmap detection demonstrated no nonspecific reactions with avian influenza virus (aiv), avian leukosis virus (alv), newcastle disease virus (ndv), and marek’s disease virus (mdv). the results also demonstrated that the xmap assay was four times more sensitive than the enzyme-linked immunosorbent assay (elisa) for iltv detection and two times more sensitive for ibv detection. a total of chicken serum samples from a chicken farm were tested by xmap and elisa assays. the results showed that the coincidence rates were . and % for iltv and ibv detection, respectively. conclusion: this study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination. infectious laryngotracheitis (ilt) and infectious bronchitis (ib) are common respiratory diseases in poultry and are currently present at epidemic levels around the world, including in china, the united states, and india. because ilt and ib have high incidence and infectivity in chickens at different ages (in days), they have caused huge economic losses to the poultry industry. both diseases have similar clinical symptoms and pathological changes, leading to significant difficulties in clinical differential diagnosis, especially in cases of mixed infection [ , ] . in addition, the use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases, such as infectious laryngotracheitis and infectious bronchitis [ ] . therefore, it is important to establish a method to simultaneously detect iltv and ibv antibodies for the differential diagnosis and immune response evaluation after vaccination. infectious laryngotracheitis virus (iltv), an alphaherpes virus, possesses at least envelope glycoprotein, two main proteins of which are the glycoprotein b (gb) and gd, respectively, which are highly conserved herpesvirus structural glycoproteins [ ] . the gd of herpes virus has an important role by binding to the host receptors [ , ] . the gd protein has been demonstrated to be a candidate antigen for recombinant vaccines [ , ] . the infectious bronchitis virus (ibv) genome encodes four major structural proteins: the spike glycoprotein (s), the membrane glycoprotein (m), the nucleocapsid (n) protein, and the envelope or small membrane protein (e) [ ] . the n protein is thought to be an appropriate diagnostic reagent for antibody detection [ ] . in this study, gd and n proteins were selected as antigen molecules for the diagnosis of ilt and ib. at present, the methods used for the diagnosis and effect evaluation of vaccine immunity of avian respiratory diseases primarily include virology detection, serological detection, and molecular biological detection. traditional methods, such as virus isolation, animal inoculation experiments, elisa, hemagglutination (ha), and hemagglutination inhibition (hi) assays are characterized by complex procedures and long periods of time required for diagnosis. despite its unique, sensitive, simple features and rapidity, polymerase chain reaction (pcr) is unable to meet the requirements for high-flux quarantine and is not favorable for the urgent screening of bulk samples. therefore, it is imperative to establish a new technology for the effect evaluation of vaccine immunity and the rapid differentiation or correct identification of important avian respiratory diseases. a new high-flux detection technology, flexible xmap (x = unknown, map = multi-analyte profiling) assay, uses variously colored polystyrene beads that is carboxylated to allow covalent coupling of protein. conjugated beads can be used to capture specific antibodies in serum, and a fluorescent secondary antibody is incubated to bind to the captured serum antibodies. a red laser is used to determine the color of the bead and a green laser to detect fluorescence intensity of bound secondary antibodies through the luminex detection system [ ] . this method is applicable for the simultaneous and rapid detection of multiplex pathogen , s antibodies, with simple operation and high accuracy that are superior to conventional methods. in this study, a method employing luminex xmap technology to simultaneously detect iltv and ibv antibodies in serum was established, optimized and used for the differential diagnosis of ibv and iltv. this assay can also be used to simultaneous monitoring of ibv and iltv antibody levels for the evaluation of immunity after vaccination. a singleplex xmap for iltv was established by testing six concentrations of the iltv gd envelope protein ( . μg, μg, μg, μg, μg, and μg per × magbeads), seen in table . the p/n value is the ratio of mfi (median fluorescence intensity) value between the positive sample and negative sample. the optimal conjugation protein concentration was . μg per × magbeads for singleplex xmap detection of iltv. the mean p/n value under the condition was . . a singleplex xmap for ibv was established by testing six concentrations of the ibv n envelope protein ( μg, μg, μg, μg, μg, and μg per × magbeads), seen in table . the p/n value is the ratio of mfi value between the positive sample and negative sample. the optimal conjugation protein concentration was μg per × magbeads for singleplex xmap detection of ibv. the mean p/n value under the condition was . . based on the results in tables and , the optimum serum dilution ratio for iltv and ibv were : and : , respectively. when singleplex xmap assay was performed. to determine the optimal serum dilition ration for simultaneous detection of iltv and ibv, a serial serum dilutions from the range of : to : were prepared and were incubated with iltv and ibv antigen conjugated microspheres. from the p/n values showed in fig. , it is obvious that the optimal serum dilution ratio for duplex xmap assay was : . the threshold is considered as the sum of the average plus three times of standard deviation, which is also named cut-off value. to determine the threshold of xmap assay for iltv and ibv, special pathogen free chicken serum samples at : dilution were used to obtain the average mfi value and standard deviation. from the table , it showed that the threshold of iltv and ibv detection were . and . , respectively. the results in figs. and demonstrated that xmap duplex assay for iltv and ibv has a high specificity since there were no cross-reactions with serum positive for other avian diseases, such as avian influenza virus intra-assay repeatability experiments showed that the coefficient of variation (cv) was . % for iltv and . % for ibv. inter-assay repeatability experiments showed that the cv was . % for iltv and . % for ibv. all cvs were less than %, indicating that the method has high repeatability. the results were shown in tables and . comparison of xmap assay with elisa using clinical samples the sensitivities of xmap and elisa were compared using : to : iltv-positive sera and : to : ibv-positive sera. the results showed that xmap detected iltv-positive sera at : and ibv-positive sera at : , while elisa detected iltv-positive sera at : and ibv-positive sera at : (see table ). to compare duplex xmap assay with elisa for iltv/ibv detection, chicken serum samples from a chicken farm in huizhou, china were used and the results are shown in table . for iltv, xmap detected positive cases whereas elisa detected positive cases; among them, samples were detected positive by xmap assay and elisa. all samples were detected as positive for ibv by both methods. ib, an acute infectious disease characterized by acute watery diarrhea, kidney necrosis, and high mortality, is caused by ibv. thus far, ib has resulted in economic losses to the poultry industry in various countries [ , ] . ibv infects chickens at different ages. chicks infected with ibv are characterized by noisy breathing, panting, sneezing, nasal fluid, and other symptoms; furthermore, laying hens exhibit significantly reduced egg production, broilers exhibits low growth and bad meat quality, and breeding hens exhibit significantly reduced breeding efficiency [ ] . ilt is an acute, contact respiratory infectious disease in chickens. chickens infected with ilt are characterized by nasal fluid, conjunctivitis, panting, coughing, and the production of bloody mucus, as well as swelling, erosion, necrosis, and extensive bleeding in the throat after analysis; laying hens exhibit reduced egg production or even no production, as well as poor egg quality [ ] . the disease spreads rapidly and has a wide distribution. the high mortality rate (> %) results in huge economic losses to the poultry industry [ ] . given the similar clinical symptoms between ilt and ib, the development of diagnoses for both diseases is significant. this is the first case report of differential diagnosis of iltv and ibv with xmap assay. luminex xmap assay is an emerging technology that uses small carboxylated polystyrene microspheres that are internally dyed with both a red and an infrared fluorophore [ ] . by changing the ratio of the two fluorophores, it is possible to distinguish up to different color-coded microsphere sets, and each microsphere set may be coupled with a different biological probe. the microspheres are detected and characterized by a dedicated flow cytometer [ ] . the luminex xmap technology is useful for many different applications. one review describes the use of this technology for the multiplex detection of viral, bacterial, parasitic, and fungal agents using the microsphere-based multiplex nucleic acid assay (mbmna) and the microsphere-based multiplex immunoassay (mbmi) [ ] . mbmis are typically biochemical tests that allow the detection or measurement of the concentration of a protein in a solution via an antibody or immunoglobulin [ ] . mbmis are often used in the diagnosis of various pathogens, such as human papillomavirus and human cytomegalovirus, to detect antibodies in serum samples [ ] [ ] [ ] . the advantages of this technology are its simple operation, high accuracy, and reduction in the number this study describes the establishment and validation of single xmap and duplex xmap assays. first, to achieve effective and rapid clinical diagnosis of ilt and ibv, single xmaps were established for the separate diagnosis of these two diseases after screening to identify the best concentrations of microsphere antigens for iltv gd and ibv n ( . μg/ × magbeads and and μg/ × magbeads, respectively), with an optimal serum dilution of : . however, it is not ideal to diagnose only one type of pathogen antibody because more than one disease often affects the respiratory tract in poultry. given the similar clinical symptoms of ilt and ibv, the differential diagnosis of these two pathogens with two single xmap assays using one sample wastes time and energy. to achieve the rapid differential diagnosis of clinical samples, it is necessary to establish a duplex xmap. in addition to iltv and ibv, aiv and ndv are also common respiratory viruses for poultry. these four diseases have high incidences, and it is difficult to make a differential diagnosis based on clinical symptoms, which are all similar. figures and showed that our xmap methods was able to simultaneously detect the antibodies to iltv or ibv in the sera and had the high specificity. a duplex xmap can quickly identify iltv and ibv infection, but it does not indicate possible infection by aiv and ndv. therefore, based on this study, we propose that quadruplex xmap should be established in the future for the detection of iltv, ibv, aiv and ndv as a fast and effective differentiation tool for these four viruses. since elisa was established to detect iltv antibodies in [ ] , it has demonstrated strong advantages, as well as higher sensitivity than virus neutralization tests (vnt), making it widely applicable. elisa antibody detection of ibv is also feasible at earlier time points [ , ] and is currently broadly applied in the poultry industry. single-analyte elisa does not support the detection of multiple specific antibody responses simultaneously for a single serum sample [ ] and possesses other disadvantages, such as the requirement for a relatively large amount of sample, negligible nonspecific binding, and increased background. however, with the development of the poultry industry, requirements dictate that many samples must be detected simultaneously; thus, a new detection method for diagnosis with iltv and ibv is significant. luminex xmap assay represents an alternative for commonly employed indirect tests such as elisa. the conversion of an elisa to the xmap format is uncomplicated, efficient and cost-saving, producing an assay with superior dynamic range and sensitivity [ ] . mbmi represents increased sensitivity and the potential to quantify antibodies, antigens, and other substances (e.g., hormones, cytokines, and tumor markers), unlike conventional elisa tests [ ] . luminex xmap has also been compared with a western blot assay for antibody detection [ ] . the results obtained from both methods showed that the sensitivity of xmap was . %, while the western blot assay sensitivity was . %. therefore, xmap may be more efficient and precise than western blots for the diagnosis of diseases. although elisa provides relatively accurate results and has been used for many years, this study demonstrates that xmap is much more sensitive than elisa for the diagnosis of ilt, as indicated by the results presented in table . in addition, xmap may be used for the rapid diagnosis of many samples, saving time and effort. the results presented in table , demonstrate that xmap is highly accurate, with similar detection results to those obtained by elisa in positive clinical samples; thus, xmap is able to be widely applicable. this study describes how to determine the optimal conditions for establishing duplex xmap assay based on the singleplex xmap result, thus providing a reference for establishing multiplex xmap assay for simultaneous detection of animal pathogen , s.antibodies. the antigens used for iltv were a recombinant gd [ ] , expressed, purified, preserved in our laboratory. for the detection of antibodies against ibv, the recombinant n protein was expressed as gst fusion protein in escherichia coli bl (de ) cells [ ] . prior to coupling, the recombinant proteins was desalted by gel filtration using microbio-spin columns (bio-rad, california, usa) based on the manufacture's protocol to exchange the buffer from sodium azide or imidazole to pbs. all the antigens were quantified using pierce bca protein quantification kit (thermo scientific, usa). coupling of proteins to fluorescent microspheres was performed as described by karanikola et al. [ ] . map mc beads (luminex, usa) were coupled by gd protein of iltv and map mc beads (luminex, usa) were coupled by n protein of ibv. a certain number of beads was transferred to one coupling reaction tube, followed by μl bead wash buffer and suspended in μl bead activation buffer. then μl of mg/ml edac (sigma-aldrich) and μl of mg/ml s-nhs (sigma-aldrich, germany) were added in the reaction buffer. the reaction tube was gently vortexed for min at room temperature (rt). pbs (ph . , μl) was added twice and the recombinant protein was added. incubation was carried the xmap assays were carried out in -well polystyrene microplates using luminex detection system (luminex, usa). relevant positive and negative sera have been prepared for the establishment of the singleplex xmap assay to detect iltv antibody or ibv antibody in the serum. iltv gd protein at the following concentrations ( . μg, μg, μg, μg, μg, and μg) and ibv n protein at the following concentrations ( μg, μg, μg, μg, μg, and μg) were coupled with × magbeads to determine the optimum antigen concentration for singleplex xmap assay. a serious dilution of sera at the range from : to : have been made to determine the optimal sample dilution. cross-reactivity or specificity was evaluated by making the assay with sera from chicken infected with aiv, alv, ndv, and mdv. then, a duplex xmap assay was established based on the result of two separated singleplex xmap assay. in briefly, a μl aliquot of the simplex beads ( beads/μl) or the duplex beads ( beads/μl, beads/μl each kind) was transferred into the wells, added in μl of diluted sera. incubation was conducted on a plate shaker ( rpm) at rt for min. the centrifugation was conducted in the magnetic separator for s. the supernatant was removed from each well. beads were washed times in μl pbs. each well was added with μl of a biotinylated secondary antibody (goat anti-chicken igy, beijing solarbio science & technology, china) at μg/ml. the plate was shaken ( rpm) for min at rt and washed as described above. finally, μg/ml of streptavidin-phycoerythrin (sape, life technologies gmbh) was added to each well at μl/well. the plate was shaken and the supernatant was removed. finally, μl of assay buffer was added to each well. the plate was shaken for approximately s and analyzed according to the manufacturer's protocol. all samples were conducted in triplicates in each assay. commercial iltv and ibv elisa kit (biochek, netherlands) were used to detect presence of iltv or ibv antibodies in poultry sera. the assay was performed according to the manufacturer's protocols. herpes virus fusion and entry: a story with many characters avian infectious bronchitis virus viral respiratory diseases (ilt, ampv infections, ib): are they ever under control? molecular biology of avian infectious laryngotracheitis virus structure of herpes simplex virus glycoprotein d bound to the human receptor nectin- antibody-induced conformational changes in herpes simplex virus glycoprotein gd reveal new targets for virus neutralization newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges coronavirus avian infectious bronchitis virus an elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications response of young rabbits to infectious bronchitis virus (wachtel strain) review of infectious bronchitis virus around the world diseases of poultry quantitative, multiplexed detection of bacterial pathogens: dna and protein applications of the luminex labmap (tm) system report from a workshop on multianalyte microsphere assays xmap technology: applications in detection of pathogens a collection of methods and protocols for developing multiplex assays with xmap technology simultaneous 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of a capture elisa to a luminex xmap assay using a multiplex antibody screening method validation and comparison of luminex multiplex cytokine analysis kits with elisa: determinations of a panel of nine cytokines in clinical sample culture supernatants luminex xmap combined with western blot improves hiv diagnostic sensitivity preparation of polyclonal antibodies against gd protein of iltv and application cloning and prokaryotic expression of nucleoprotein gene of infectious bronchitis virus (ibv) zz strain development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against cooperia oncophora, dictyocaulus viviparus and fasciola hepatica in cattle availability of data and materials all data generated or analyzed during this study are included in this published article. contact corresponding author for requests. all authors contributed to the interpretation of the data, critically revised the manuscript for important intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work.ethics approval and consent to participate not applicable. our manuscript does not contain any individual person's data in any form. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -dwok k authors: li, heng; li, hongzhe; wang, jingjing; guo, lei; fan, haitao; zheng, huiwen; yang, zening; huang, xing; chu, manman; yang, fengmei; he, zhanlong; li, nan; yang, jinxi; wu, qiongwen; shi, haijing; liu, longding title: the altered gut virome community in rhesus monkeys is correlated with the gut bacterial microbiome and associated metabolites date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: dwok k background: the gut microbiome is closely associated with the health of the host; although the interaction between the bacterial microbiome and the whole virome has rarely been studied, it is likely of medical importance. examination of the interactions between the gut bacterial microbiome and virome of rhesus monkey would significantly contribute to revealing the gut microbiome composition. methods: here, we conducted a metagenomic analysis of the gut microbiome of rhesus monkeys in a longitudinal cohort treated with an antibiotic cocktail, and we documented the interactions between the bacterial microbiome and virome. the depletion of viral populations was confirmed at the species level by real-time pcr. we also detected changes in the gut metabolome by gc-ms and lc-ms. results: a majority of bacteria were depleted after treatment with antibiotics, and the shannon diversity index decreased from . to . . furthermore, the abundance-based coverage estimator (ace) decreased from . to . , and the abundance of eukaryotic viruses also changed substantially. in the annotation, families of dna viruses and bacteriophage family were present in the normal monkeys but absent after gut bacterial microbiome depletion. intriguingly, we discovered that changes in the gut bacterial microbiome composition may promote changes in the gut virome composition, and tryptophan, arginine, and quinone may play roles in the interaction between the bacterial microbiome and virome. conclusion: our results indicated that the clearly altered composition of the virome was correlated with depletion in the bacterial community and that metabolites produced by bacteria possibly play important roles in the interaction. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. the gut microbiome of an animal consists of bacteria, viruses, fungi and so on. this intricate ecosystem interacts with the adjacent epithelial layer, and the microbes perform metabolic functions, protect against pathogens, and condition the immune system, and through these basic functions, these microbes directly or indirectly affect most of the physiological functions of the host [ , ] . in recent years, variations in the bacterial community composition have been shown to correlate with infection outcome, inflammatory bowel disease [ ] , diabetes [ ] , obesity [ ] , and depression [ ] , and fecal microbiome transplantation has become an effective treatment for refractory clostridium difficile infections and other diseases [ , ] . the mechanisms of interaction between the gut bacterial microbiome and the host are very complex, and other components also play crucial roles in this process. in addition to bacteria, viruses are also abundant in the gut [ ] and have been hypothesized to markedly alter the structure and function of the bacterial community [ ] [ ] [ ] . additionally, chronic viral infection can confer increased resistance against pathogenic challenges [ ] . gut virome alteration has been observed in inflammatory diseases such as inflammatory bowel disease and crohn's disease [ ] . the recent advent of highthroughput sequencing methods has made it possible to study these communities and their relationships with health and disease in detail [ ] . bacterial communities play an essential role in host health, but further research is still warranted to obtain an in-depth understanding of the mechanisms underlying this role. transfer of whole virome communities between humans was documented in fecal microbiome transplantation [ ] , and the difference varied more widely between gut viromes than between gut bacterial microbiomes in humans [ ] . however, the relationship between the bacterial microbiome and the virome has rarely been studied, despite its likely medical importance. previous research has shown close relationships between single viral species and single bacterial species [ , ] , and single viral species could trigger shifts in the bacterial microbiome and the virome [ , ] . at the same time, enteric bacteria were seen to be required for efficient infection by [ , ] or suppression of [ ] viruses, and the richness of the gut bacterial microbiome had an obvious effect on bacteriophage composition [ ] ; moreover, the gut virome composition in humans was examined, and bacteriophage diversity was found to be inversely correlated with naturally occurring bacterial diversity in human infants during healthy development [ ] . however, few studies have focused on how the whole virome, a diverse community consisting of eukaryotic rna and dna viruses and bacteriophages, interacts with the bacterial microbiome. rhesus monkeys are good mammalian research models that are closely related to humans, and the virome composition of these animals was seen to be affected by simian immunodeficiency virus infection [ ] . we hypothesized that there is a close relationship between the whole gut virome and bacterial microbiome, and the bacterial microbiome could be depleted by treatment with an antibiotic cocktail in rhesus monkeys. we then examined the virome composition to detect the direct effects of the bacterial microbiota on the virome. we performed s rrna amplicon sequencing of the fecal bacteria and metagenomic analysis of fecal viromes from rhesus monkeys treated with an antibiotic cocktail. our results suggest that a majority of bacteria were depleted after the monkeys were treated with antibiotics and that the composition of the whole virome changed drastically. importantly, alteration of the virome along with shifts in the composition and function of the gut bacterial community and metabolites from gut bacteria may have played an important role in the interaction. the rhesus monkey cohort described in this study was housed at the institute of medical biology, chinese academy of medical sciences (imbcams). an antibiotic cocktail containing ampicillin, streptomycin, kanamycin, metronidazole, and vancomycin was administered orally at a dose of mg/ kg times per day for weeks. three healthy one-year-old rhesus monkeys were treated with antibiotics, and fresh fecal samples were collected one day before treatment with antibiotics and , , and days after treatment with antibiotics and stored at − °c for subsequent analysis. fresh fecal samples from an additional three normal monkeys were collected after the monkeys were treated with antibiotics for days. the bacterial community of each sample was detected by s rrna amplicon sequencing, and the virome communities in the samples collected before treatment with antibiotics and in those collected after treatment with antibiotics for days were detected by deep sequencing. because metabolome analysis requires biological duplications, the metabolomes of samples collected before treatment with antibiotics and of those collected after treatment with antibiotics for and days were detected by gc-ms and lc-ms. in our analysis, samples collected before treatment with antibiotics were used as the control group, and samples collected after treatment with antibiotics were used as the experimental group. bacterial s rrna amplicon sequencing dna was extracted from fecal samples, and pcr was performed with the barcode primers f/ r to obtain amplicons of hypervariable regions v and v for phylogenetic discrimination analysis [ ] . libraries were pooled by using a truseqtm dna sample prep kit and sequenced using an illumina miseq sequencer. sequences were assigned to closed-reference operational taxonomic units (otus) at a % identity threshold using bacterial s rrna amplicon sequences from the silva / sbacteria database. the otu data were rarefied to the smallest effective sample sizes [ ] ; rarefaction is a homogenization method that is used to randomly draw otus to the same quantity based on a minimum value. the α diversity, which includes the abundance-based coverage estimator (ace) and shannon diversity index, was analyzed by mothur (version v. . . ), and statistical significance was evaluated by student's t-test. kyoto encyclopedia of genes and genomes (kegg) prediction analysis of the bacterial microbiome [ , ] we performed a kyoto encyclopedia of genes and genomes (kegg) prediction analysis of the bacterial microbiome using picrust. picrust contains the cluster of orthologous groups of proteins (cog) and kegg ortholog (ko) information corresponding to greengene id numbers. for metagenome prediction, picrust takes an input otu table containing identifiers that match tips from the marker gene with corresponding abundances for each of the otus across one or more samples. first, picrust normalizes the otu table based on s rrna amplicon copy number prediction so that the otu abundances accurately reflect the true abundances of the underlying organisms. the metagenome is then predicted by looking up the precalculated genome content for each otu, multiplying the normalized otu abundance by each ko abundance in the genome and summing these ko abundances together per sample. the prediction yields a table of ko abundances for each metagenome sample in the otu table. analysis of similarities (anosim) is a nonparametric test that shows whether the difference between groups is greater than that within groups. the analyses were performed in vegan or qiime in r (version . . ) by using the bray-curtis algorithm [ ] . fecal samples were suspended in phosphate-buffered saline (pbs) and filtered through a filter with a pore size of . μm (millipore). the supernatant was enriched by a -kda molecular mass filter (ultra- k, millipore). the concentrate was treated with dnase i (takara) at °c for min to eliminate unencapsulated nucleic acids. subsequently, total viral dna was extracted from half of the concentrate using the qiaamp dna stool kit (qiagen), and at the same time, total viral rna was extracted from the other half using the qiaamp viral rna kit (qiagen). the extracted rna was synthesized into double strands using the nebnext rna first strand synthesis module (neb) and the nebnext mrna second strand synthesis module (neb). the dna and double-stranded cdna were amplified by whole-genome amplification (repli-g mini kit, qiagen) and then fragmented into approximately -bp fragments by a covaris m instrument. then, the fragments were amplified into a pe library by the truseq™ dna sample prep kit and fixed to the chip by bridge pcr using the hiseq / pe cluster kit. the constructs were sequenced on the illumina hiseq platform using hiseq / sbs kits. for virome analysis, we first sheared the adaptor sequences with seqprep and removed reads that were shorter than bp and those that contained n bases with sickle to retain the paired-end reads and single-end reads. we compared these reads to the host (rhesus monkey) genome by bwa and removed the reads belonging to the host. then, we compared all the clean reads with the u.s. national center for biotechnology information (ncbi) nucleotide database to identify the sequences that belonged to viruses and the sequences that did not belong to any known genome, such as those of bacteria, fungi or other known microorganisms. then, contigs were built from these reads. the contigs and all reads that could not be mapped to any known genome in ncbi were compared with the virus protein database in the ncbi nonredundant refseq database (including sequences from swissprot, pir, prf, and pdb and coding sequences (cds) from genbank and refseq) based on amino acid sequences using blastp (blast version . . +, e-value: e- ). these results constituted our virus database and were used to obtain the nonredundant gene catalog by cd-hit. all the reads were compared to our virus database to analyze their richness. the spliced read alignments were predicted by meta-gene, compared to the eggnog database and virulence factors database (vfdb) for cog analysis using blastp (blast version . . +, e-value: e- ) and annotated using vfdb. the abundance results that were similar in or more monkeys were selected, and real-time pcr was used to validate the changes in these viruses (sybr premix ex taq ii, takara). as the template, we used the dna and cdna extracted from fecal samples. the samples that were not detected directly from the dna or cdna were subjected to multiple displacement amplification (mda) (total nucleic acid was amplified by multiple displacement to comprehensively detect both dna and rna viruses [ ] ) and then analyzed by real-time pcr. for real-time pcr, we used the ct numbers to show the richness of the virus. viruses that were not detected were not shown. primers were designed to amplify specific regions in the bdellovibrio phage phimh k ( ′-aatcctcaattccagacttcca- ′ (f) and ′-ccat ttccataagtccgagtg- ′ (r)), bacillus phage b ( ′-tggcgatgttgatgatgac- ′ (f) and ′-cttt atttgcgtctgttgtcg- ′ (r)), columbid circovirus ( ′-tcaggagacgaaggacacg- ′ (f) and ′-tggc atcatacatcgggac- ′ (r)), potato virus m ( ′-cgct tcgctgctttcg − ′ (f) and ′-cggaccattcatac cacca- ′ (r)), marseillevirus marseillevirus ( ′-aaagtc ccaagttatcacaagc- ′ (f) and ′-tttctcgcag cgtcaatg- ′ (r)), simian sapelovirus ( ′-ttccatct gctctaaatgctca- ′ (f) and ′-cagcagttagag cgggtg- ′ (r)), and andean potato mild mosaic virus ( ′-aagcccaacatcgttctcc- ′ (f) and ′-aaga ggatacgggagaaagg- ′ (r)). redundancy analysis (rda) shows the interactions between sample distribution and environmental factors. we used vegan's rda analysis in r with the phylumlevel abundances of the bacterial microbiome as environmental factors. we ran a regression analysis between bacterial microbiome diversity and virome richness with the stats package and plotted the results using the ggplot package. the stool samples were suspended in methanol:h o ( : ), ground, ultrasonicated, concentrated and dried so that the metabolome could be analyzed by gc-ms and lc-ms. the derivatized samples were analyzed on an agilent a gas chromatography system coupled to an agilent c msd system (agilent). an hp- ms fusedsilica capillary column ( mm × . mm × . μm, agilent) was utilized to separate the derivatives. helium (> . %) was used as the carrier gas at a constant flow rate of . ml/min through the column. the injector temperature was maintained at °c. a volume of μl was injected in splitless mode. the oven temperature was initially °c and was then ramped up to °c at a rate of °c/min, to °c at a rate of °c/min, to °c at a rate of °c/min and to °c at a rate of °c/min; finally, the temperature was held at °c for . min. the temperatures of the ms quadrupole and ion source (electron impact) were set to °c and °c, respectively. the collision energy was ev. mass data were acquired in full-scan mode (m/z - ), and the solvent delay time was set to min. the acquired ms data from gc-ms were analyzed by chromatof software (v . , leco, st joseph, mi). metabolites were qualitatively assessed by the fiehn database, which is linked to chromatof software. briefly, after alignment with the statistic compare component, a csv file was obtained with three-dimensional data sets, including sample information, peak name, retention time, m/z and peak intensities. the resulting data were normalized to the total peak area of each sample in excel (microsoft, usa) and imported into simca (version . , umetrics, umeå, sweden) to define the % confidence interval of the modeled variation. the differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (vip) values obtained from the opls-da model and p values from a two-tailed student's t-test on the normalized peak areas, where metabolites with vip values larger than . and p values less than . were included. lc-ms was performed on an ultimate -velos pro system equipped with a binary solvent delivery manager and a sample manager coupled with an ltq orbitrap mass spectrometer equipped with an electrospray interface (thermo fisher scientific); an acquity beh c column ( mm × . mm i.d., . μm; waters) was used. the column was maintained at °c, and separation was achieved using the following gradient: % b- % b from to . min, % b- % b from . to . min, % b from . to . min; % b- % b from . to . min, and % b from . to . min at a flow rate of . ml/min, where b was acetonitrile ( . % (v/v) formic acid), and a was aqueous formic acid ( . % (v/v) formic acid). the injection volume was . μl, and the column temperature was set at . °c. the mass spectrometric data were collected using an ltq orbitrap mass spectrometer equipped with an electrospray ionization (esi) source operating in either positive or negative ion mode. the capillary and source temperatures were set at °c, with a desolvation gas flow of l/h. centroid data were collected from to m/z with a resolution of , . xcms (http://masspec.scripps.edu/ xcms/xcms.php) was used for nonlinear alignment of time domain data and automatic integration and extraction of the peak intensities. default xcms parameter settings were used (major default parameters: profmethod = bin; method = matched filter; step = . ) except for full width at half maximum = , bandwidth (bw) = and snthresh = . variables with < % relative standard deviation (rsd) in qc samples were then retained for further multivariate data analysis. the result was a three-dimensional matrix that included retention time and m/z pairs (variable indices), sample names (observations), and normalized ion intensities (variables). the positive and negative data were merged into a combined data set, which was imported into simca-p+ . software (umetrics, umeå, sweden). the differential metabolites were selected on the basis of a combination of statistically significant vip values obtained from the opls-da model and p values from a two-tailed student's t-test on the normalized peak areas, where metabolites with vip values larger than . and p values less than . were included. the differential metabolites were qualitatively assessed using the human metabolome database (http://www.hmdb.ca/) and metlin (https://metlin. scripps.edu/). we performed metagenomic sequencing of fecal samples to detect the bacterial microbiome and virome composition of healthy one-year-old rhesus monkeys housed at the imbcams (fig. ) . the rhesus monkeys were monitored by blood cell analysis, which is the examination of blood condition and disease by observing the number and distribution of blood cells during the course of antibiotic treatment [ ] , and we found no obvious differences between the normal monkeys and those treated with antibiotics (additional file : figure s ). the composition of the bacterial microbiome was investigated by extracting dna directly from feces for s rrna gene amplification. we used the hypervariable regions v and v to perform phylogenetic discrimination with the barcode primers f/ r [ ] . in total, , amplicon reads ( , ± per sample) were obtained. at the phylum level, the fecal bacterial communities were composed predominantly of high abundances of bacteroidetes ( . %) and firmicutes ( %) and low levels of proteobacteria ( . %) (additional file : figure s a ). as expected, the enteric bacterial microbiome was depleted significantly after exposure to antibiotics (fig. , a ; additional file : figure s , additional file : table s , additional file : figure s ). the levels of firmicutes and bacteroidetes decreased to . % and below . %, respectively; at the same time, the abundance of proteobacteria increased to % (additional file : figure s b ). escherichia-shigella were the major constituents of proteobacteria, and other genera belonging to proteobacteria were markedly depleted (additional file : figure s b , additional file : table s ). the fecal samples were spread on plates with the antibiotic cocktail, and a large number of colony-forming units (cfus) were observed in the sample from d but not in the sample from d . the s rrna amplicons of single clones were sequenced, and we found that these clones belonged to escherichia-shigella (data not shown). as expected, the overall diversity and richness of the bacterial microbiome were depleted both stably and continuously (fig. b, c) . the shannon diversity index showed that the diversity of the bacterial microbiome was significantly decreased after treatment with antibiotics and remained at a low level (fig. b , additional file : figure s c ). the richness of the bacterial microbiome was significantly decreased, as measured by the ace (fig. c , additional file : figure s d ). in addition, fig. experimental procedure. three rhesus monkeys were treated with an antibiotic cocktail to control their gut bacterial microbiome, and we detected the longitudinal changes in the gut bacterial microbiome at d , d and d by s rrna amplicon sequencing. then, we extracted nucleic acids from the fecal supernatant at d and d and scanned the gut viromes of the monkeys. the samples for metabolomics were collected on d , d and d and scanned by gc-ms and lc-ms. we comprehensively analyzed the interactions among the gut virome, bacterial microbiome and metabolomes based on the above results there were noticeable differences in bacterial β-diversity between control and experimental animals, as determined using principal component analysis (pca), and the results showed good repeatability within a single group (additional file : figure s f ). together, these data suggest that the richness and diversity of the bacterial microbiome composition were depleted stably and continuously. therefore, we assessed the virome composition in the control and antibiotictreated experimental monkeys on the ninth day. in previous experiments, examination of microbiota genomes from rhesus macaques (macaca mulatta) showed that a majority of the sequences in the fecal samples were mapped to bacterial genomes, while the percentage of sequences mapped to viral genomes was very low [ ] . to comprehensively detect both dna and rna viruses, we filtered the fecal samples with a . -μm filter and treated the samples with dnase i, after which, total dna and rna were extracted separately from the same fecal sample. owing to low yield, amplification of the dna and double-stranded cdna by whole-genome amplification (using mda) was necessary. using the miseq × paired-end protocol on the illumina miseq platform, we obtained an average of , , ± , , clean reads per mda sample library and generated a total of , mbp from samples, allowing detailed investigation of the viral populations. to catalog the present genes, we predicted open reading frames (orfs). a total of , orfs were predicted; the average orf length was . bp, with a maximum of , bp and a minimum of bp. the fig. the bacterial microbiome was obviously depleted by treatment with antibiotics. heatmap of the out percentage of every genus at timepoints. each point represents biological replicates. the genera that belong to the same phylum are shown in the same color on the left. the total otu number of biological replicates for every genus was more than . the color bar represents the log of the percentage, the numbers in the heatmap are the log values of the otu numbers, and the numbers in the bar are the percentages ecological signatures of the intestinal virome have been characterized. the largest percentage of sequences mapped to viromes in the fecal samples belonged to bacteriophages, accounting for over % of the sequences (additional file : table s ). we also identified eukaryotic dna viruses and rna viruses, as well as other viral families that are defined as "unclassified" in the ncbi taxonomy database. in the raw data from the virome metagenomic analysis, rna virus sequences were found in the dna virus results, and dna virus sequences were found in the rna virus results, because the rna virus sequences were wrongly affiliated with dna viral genomes and vice versa. in addition, dna viruses, especially bacteriophages, were the main components of the virome in our sequencing data, and the rna from these viruses may have been extracted together with rna viruses and vice versa. thus, we excluded these data. we observed that the fecal virome composition was noticeably altered after depletion of the bacterial microbiome treated with antibiotics. anosim showed that the distance between groups was greater than that within groups for dna viruses and bacteriophages (additional file : figure s ). dna viruses, including members of the families poxviridae, iridoviridae, ascoviridae, baculoviridae, marseilleviridae, and mimiviridae and bacteriophages, such as members of the family inoviridae, were present in the normal monkeys but absent after the gut bacterial microbiome was depleted (fig. ) . dna viruses, including members of the families herpesviridae, nanoviridae, and phycodnaviridae, were present in three biological replicates before antibiotic treatment but in only one biological replicate after the gut bacterial microbiome was depleted (fig. ) . most of the reads from bacteriophages were noticeably depleted after the gut bacterial microbiome was depleted (additional file : figure s , additional file : table s ). rna viruses, including members of the families picornaviridae and tymoviridae, were present in three biological replicates but were present in only one biological replicate after the gut bacterial microbiome was depleted, and rna viruses belonging to nodaviridae were present in two biological replicates but absent after the gut bacterial microbiome was depleted (fig. ). in addition, many kinds of viral groups, including circoviridae, geminiviridae, microviridae, podoviridae, myoviridae, siphoviridae, picornaviridae, and retroviridae, were present regardless of whether the bacterial microbiota was depleted, but the sequencing reads showed that the abundances of these viruses may have decreased with bacterial microbiota depletion (additional file : figure s ). however, we could not validate this decrease regarding the abundances of viruses at the species level, the results that were similar in or more monkeys were selected, and we used real-time pcr to validate changes in the richness of the viruses. as the figure shows, we analyzed fecal samples that had not been subjected to mda. after the bacterial microbiome was depleted, the results from real-time pcr validated the depletion of viral species: bdellovibrio phage phimh k, which belongs to the microviridae family of bacteriophages; bacillus phage b , which belongs to the podoviridae family of bacteriophages; columbid circovirus, which belongs to the circoviridae family of dna viruses; and potato virus m, which belongs to the betaflexiviridae family of rna viruses (fig. a) . the samples that were not detected directly from dna or cdna were subjected to mda and then detected by real-time pcr. the depletion of viral species was detected (fig. b) : marseillevirus marseillevirus, which belongs to the marseilleviridae family of dna viruses; simian sapelovirus, which belongs to the picornaviridae family of rna viruses; and andean potato mild mosaic virus, which belongs to the tymoviridae family of rna viruses. in addition, mason-pfizer monkey virus, which belongs to the retroviridae family, was detected among the rna viruses; this virus is very dangerous in monkey populations and has been shown to cause an aids-like disease in rhesus macaques [ ] . encouragingly, no mason-pfizer monkey virus was detected by real-time pcr. briefly, the fecal virome composition was noticeably altered after depletion of the bacterial microbiome, and the abundances of many dna viruses, bacteriophages and rna viruses in the gut were clearly decreased. in addition, in the metagenomic analysis, we found high numbers of reads from dna viruses and bacteriophages; however, low numbers of reads from rna viruses were found (additional file : figure s , additional file : table s ). these results may be due to the limited application of mda in rna viruses. the microbiota structure is the result of dynamic interactions among various community members. we found fig. the composition of the virome changed noticeably after treatment with antibiotics. the presence-absence heatmap shows the virome characterized by metagenomic analysis. due to the presence of low-complexity/repetitive regions in the reads, false-positive virus family taxonomic assignments with fewer than reads were omitted from the analyses [ ] a close interaction between the whole virome (dna viruses, rna viruses and bacteriophages) and the bacterial microbiome. we analyzed the effects on the abundance of the virome by rda, taking the richness of bacteria at the phylum level as environmental factors, and found a negative interaction between the abundances of dna viruses and bacteriophages at d and the abundances of most bacteria (fig. a, b) . however, rna viruses exhibited chaotic interactions due to weak repeatability. next, we conducted linear regression analysis between virome abundance and both the ace and shannon diversity index, and we found positive correlations in bacteriophages (fig. c, d) . in addition, the dna and rna viruses showed a positive trend (additional file : figure s ) but weak confidence levels. overall, these results support our hypothesis that a clear interomic relationship exists between the virome and bacterial microbiome. positive correlations were found between virome abundance and the richness and diversity of the bacterial microbiome. metabolites could inhibit or promote viruses in vivo [ ] and in vitro [ ] [ ] [ ] , and the metabolites produced by bacteria play important roles in host physiology [ ] . to interrogate the functions associated with the response to depletion in the bacterial microbiome, we performed a kegg prediction analysis of the bacterial microbiome using picrust. we observed significant differences in functional systems along with shifts in the composition of the bacterial microbiome, according to the predictions by picrust. kegg pathways associated with bacterial toxins were downregulated significantly (fig. a) , perhaps as a result of antibiotic treatment. the pathway associated with d-arginine metabolism showed a -fold decrease; in contrast, the pathways associated with fatty acid metabolism and tryptophan metabolism showed a -fold and . -fold increase, respectively. at the same time, the biosynthetic pathway for ubiquinone and other terpenoidquinones showed a -fold increase. moreover, the glycosaminoglycan degradation pathway exhibited low diversity and a -fold decrease (fig. a) . glycan [ ] , glycosaminoglycan [ ] , quinone [ ] and arginine [ , ] are well known to support the inhibition of viruses, while tryptophan [ , ] and fatty acids [ ] promote viral survival. interestingly, we detected the metabolites in the fecal samples by metabolome scanning and found that the changes in some metabolite levels were consistent with our prediction based on the normalization of otus in the s rrna amplicon sequencing data. because metabolomic analysis requires biological duplications, the metabolomes of samples collected before treatment with antibiotics and those of samples collected after treatment with antibiotics for and days were examined by gc-ms and lc-ms. the metabolomic results exhibited good repeatability (additional file : figure s ). in gc-ms (fig. b) , n-dimethylarginine was not detected in monkeys that were not treated with antibiotics, but the metabolite was present after antibiotic treatment. the levels of n-acetyltryptophan and n-methyltryptophan showed a . -fold and . -fold decrease, respectively, after treatment with antibiotics. in lc-ms (fig. c) , we found that the levels of abruquinone b and sulfated dihydromenaquinone- exhibited a -fold and . fold increase, respectively. regrettably, in the present study, we did not measure the levels of glycosaminoglycan, which plays a very important role in reducing the viral population [ ] . as reported by adina howe, yatsunenko t and alejandro reyes, in the same environment and feeding conditions, the composition of the microbiota and virome could remain stable within an individual [ , , ] . however, the gut microbial composition could be influenced by multiple interacting factors, such as diet [ ] , antibiotic use [ ] , age, geographical setting [ ] , and several diseases, including chronic inflammation, obesity and diabetes [ ] . in our study, the major reason for depletion of the gut bacterial microbiota was treatment with the antibiotic cocktail. the feeding conditions of the rhesus monkeys were stable in terms of their food and water consumption, and blood samples were monitored routinely, showing that there was no infection during the study period (additional file : figure s ). the bacterial composition exhibited stable and continuous depletion after treatment with the antibiotic cocktail, and we found that the virome composition changed noticeably and was correlated with the shifts in the bacterial community. moreover, we found that metabolites produced by the gut bacterial microbiome may play a role in the interrelation. in addition, we found that the composition of the rhesus monkey enterovirus group was similar to that of the human enterovirus group [ ] , and our results may be beneficial for research on the composition of the human virome. when the bacterial microbiome was depleted, ampicillin could kill most bacteria, including gram-positive and gram-negative bacteria; streptomycin could kill most bacilli; kanamycin could kill most gram-negative bacteria; metronidazole could kill most anaerobic bacteria and parasites; and vancomycin could kill most gram-positive bacteria. of course, the numbers of drug-resistant bacteria are increasing, but we believe that the cocktail of five antibiotics could deplete most of the commensal bacteria in the gut. as expected, the whole gut bacterial microbiome, including gram-positive and gram-negative bacteria (additional file : figure s e ), was depleted after treatment with the antibiotic cocktail, except for escherichia-shigella species belonging to proteobacteria, which were resistant to the cocktail. escherichia harbored the most diverse antibiotic resistance genes, including genes resistant to multidrug treatments, tetracycline, aminoglycoside, macrolide-lincosamidestreptogramin b, β-lactams, and sulfonamides [ ] . we maintained the bacteria belonging to escherichia-shigella in plates with the antibiotic cocktail. in the future, we will investigate the specific resistance and antibiotic resistance genes in this bacterium. notably, our study focused on the interaction between virome composition and the bacterial microbiome in rhesus monkeys and may serve as a model for gut microbiota analysis. therefore, we used the administration of distinct antibiotics at high dosages and high frequencies for weeks to deplete the whole gut bacterial microbiome. in our results, the richness and diversity of the bacterial community were depleted. because our study did not involve clinical treatment, the normal dose of antibiotics was not evaluated by our procedure. people are widely prescribed antibiotics each year [ ] , and while antibiotics exert very complex effects on the whole bacterial microbiome [ ] , the effects of these drugs on the virome are not clear. antibiotics can directly affect viruses but do not exhibit a wide range of roles. minocycline [ ] , berberine, abamectin, ivermectin [ ] , glycopeptides [ ] , and teicoplanin [ ] could inhibit the corresponding viruses. in our study design, an antibiotic cocktail that included ampicillin, streptomycin, kanamycin, metronidazole, and vancomycin was administered orally. no study has yet reported that these antibiotics directly affect viruses. based on our results, the richness of these viruses was very low in the gut, and we had to use mda to perform deep sequencing, although the detection by deep sequencing was very sensitive. we first characterized the shift in the gut virome by deep sequencing, and the samples were amplified by mda. mda is used as a general technique in virome research, especially for dna virome detection [ ] . to a certain extent, the amplification read-out can also represent the virus quantity. however, mda is not well suited to the detection of rna viruses. the sequence-independent amplification (sia) approach is more appropriate than mda for detecting rna viruses [ ] . in the future, we can use this approach to precisely detect rna viruses. in this case, we validated the depletion of the virome composition, including dna viruses, rna viruses and bacteriophages, by real-time pcr. although the number of cycles seemed high, these results were verified via three biological replicates, and the results of the no-template control (ntc) were not detected. in addition, we performed serial dilution of the in vitro transcribed rna of coxsackievirus a to generate a standard curve and found that a ct of . represents genomic copies (data not shown). in our opinion, these viruses are components of the gut microbiome with low richness and may be involved in host physiology. the metabolites produced by gut bacteria play very important roles in host physiology [ ] , although the effects of these metabolites on virome composition have rarely been reported. glycan [ ] , glycosaminoglycan [ ] , quinone [ ] and arginine [ , ] support the inhibition of viruses, while tryptophan [ , ] and fatty acids [ ] promote viral survival. although most metabolites that can inhibit or promote viruses play roles in human viruses, tryptophan could promote the simian immunodeficiency virus in macaques [ ] . in addition, most pandemics originating in animals, such as severe acute respiratory syndrome and pandemic influenza, could start to appear because of ecological, behavioral, or socioeconomic changes [ ] . many human viruses are zoonotic, and some human viruses, such as human enterovirus , can infect animals, especially monkeys [ ] . we believe that metabolites play roles in a broad spectrum of viruses and that changes in the metabolites may correlate with depletion of the virome. in our results, the level of quinone, which decreases the abundance of viruses, was increased in the gut metabolome, and the levels of some amino acids that promote the survival of viruses, such as tryptophan, were decreased. importantly, glycosaminoglycan, which can reduce the populations of various viruses, was noticeably increased in the kegg pathways of the bacterial microbiome, but we did not measure glycosaminoglycan levels in the present study. it is very difficult to detect glycosaminoglycan by metabolic scanning because glycosaminoglycan has a very high molecular weight. in the future, glycosaminoglycan levels could be measured by time-offlight mass spectrometry. first, the polysaccharide needs to be dispelled, followed by detection of the monosaccharide to calculate the polysaccharide levels based on the relationships among the monosaccharides in a specific database. however, this process is very complicated, and the database is not sufficiently large at present. by analyzing the relevant data, we found that depletion of bacteria directly promoted changes in the concentrations of some metabolites, which may play important roles in reducing the abundance of dna viruses. our metagenomic-scale characterization of the virome composition after treatment with antibiotics supports the notion that the composition of the virome is noticeably altered in correlation with bacterial community depletion and that metabolites produced by bacteria possibly play important roles in the interaction. the next step will be to investigate the underlying mechanisms in detail. additional file : figure s . the detection of blood cell analysis during the course of antibiotic treatment. white blood cells (wbc), neutrophilic granulocytes (neut), lymphocytes (lymph), monocytes (mono#), eosinophils (eo#), and basophilic granulocytes (baso#) were counted, and the counts were compared between the monkeys that were treated with antibiotics and ones were not, and there was no obvious difference. (tif kb) additional file : figure s . the richness and diversity of gut bacterial microbiota were depleted obviously stably and continuously. (a, b) the community analysis of gut bacterial microbiota on phylum level. the phylum was represented by own color.(c, d) the student's t-test of alpha diversity index (the shannon diversity index and the ace estimator) in genus level. . < p ≤ . was marked *, . < p ≤ . was marked * *, p ≤ . was marked * * * .(e)the longitudinally reads of otu in grampositive and gram-negative bacteria. (f) the repeatability analysis of s rrna amplicon sequencing by pca. (tif kb) additional file : table s . the reads number on the genus level in the gut bacterial communities in a longitudinal cohort treated with an antibiotic cocktail. 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(tif kb) additional file : table s . the reads number on the family level in the gut virome communities in a longitudinal cohort treated with an antibiotic cocktail. (xls kb) additional file : figure s . the anosim analysis of virome groups. the ordinate represents the distance value. r value represents the statistic results, and the closer the r value is to , the greater the difference between groups than the difference in the group, and the grouping was reasonable. (tif kb) additional file : figure s all data generated or analyzed during this study are included in this published article and the additional files. we have deposited the raw sequencing data into ncbi and the number is prjna .ethics approval and consent to participate all animal experiments were conducted under prior approval from the animal ethics committee of the institute of medical biology, chinese academy of medical sciences, with permit number dwsp , according to the national guidelines on animal work in china. not applicable. the authors declare that they have no competing interests.author details key: cord- -cko curf authors: cheng, han; koning, katie; o’hearn, aileen; wang, minxiu; rumschlag-booms, emily; varhegyi, elizabeth; rong, lijun title: a parallel genome-wide rnai screening strategy to identify host proteins important for entry of marburg virus and h n influenza virus date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: cko curf background: genome-wide rnai screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. however, discrepancies on target identification of the same viruses by different groups suggest that high throughput rnai screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. methods: two genome-wide rnai screens were performed in parallel against the entry of pseudotyped marburg viruses and avian influenza virus h n utilizing an hiv- based surrogate system, to identify host factors which are important for virus entry. a comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. results: the parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. the power of this strategy is illustrated by a genome-wide rnai screen for identifying the host factors important for marburg virus and/or avian influenza virus h n as described in this study. conclusions: this strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level (bsl- ) containment instead of the bsl- or bsl- for the infectious viruses, with alleviated safety concerns. the screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate. skepticism on the utility of this technology. thus, to avoid potential screening artifacts and other issues, it is prudent to carefully design high throughput rnai screening strategies and to optimize the parameters in the pilot experiments prior to the large-scale screens. another obstacle in working with highly pathogenic viruses is that they require high containment facilities (biosafety level- or , or bsl- or bsl- ), which are not readily available for many researchers. however this problem can be often circumvented by an hiv- based surrogate system in the entry studies and in the drug discovery efforts to identify and develop antivirals targeting the entry process [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this hiv- based surrogate assay is particularly amenable for high throughput screens because it is safe and robust. in this report, we describe a genome-wide rnai high throughput screen strategy, referred to as parallel genome-wide rnai screen, which allows us to quickly identify host factors important for the entry process of highly pathogenic viruses. this strategy was used for an rnai screen to identify host proteins specific for the entry process of marburg virus (marv) or avian influenza virus h n (aiv), demonstrating the utility of this approach. to perform a high throughput rnai screen to identify the host factors involved in viral entry of highly pathogenic marburg virus and avian influenza virus h n , which require bsl- and bsl- facilities, respectively, we adopted a surrogate system which allows us to perform the initial screening in a bsl- facility [ ] . this human immunodeficiency virus- (hiv- ) based surrogate assay has been widely used by us and others to investigate the entry mechanisms of highly pathogenic enveloped viruses such as filoviruses [ , , ] (ebola and marburg viruses), avian influenza virus h n [ ] , and severe acute respiratory syndrome coronavirus (sars-cov) [ ] . it has also been used to identify and develop entry inhibitors as antivirals [ , ] . in this study, avian influenza virus h n pseudovirions (aiv [ ] ) and marburg virus pseudovirions (marv [ ] ) were generated and used in the genome wide rnai screen, as described in the materials and methods. the basic principle of this surrogate assay is based on the following two aspects: ( ) aiv and marv viral envelope glycoproteins (ha and gp, respectively) are necessary and sufficient to mediate virus entry, and ( ) these glycoproteins can be efficiently incorporated into hiv- viral particles. therefore these pseudovirions retain the entry property dictated by avian influenza virus h n glycoprotein (ha) or marburg glycoprotein (gp). thus, aspects of these entry mechanisms can be evaluated despite of the surrogate nature of the viral particles. furthermore, the pseudovirions carry a luciferase reporter gene which can be used to measure the entry activity of aiv and marv [ ] . the overall rnai screening protocol is outlined in fig. . the key feature of the protocol is that aiv and sirna showing low signals in assay plates of both viruses is regarded as a "shared" hit by the two viruses marv pseudovirions were used in parallel in the rnai screen which allowed us to reduce the number of false positives and to quickly identify marburg-specific and flu-specific host factors (which will be further discussed below). briefly, human a cells (~ cells/well) were reverse transfected with nm small interfering rnas (sirnas) in two identical -well plates simultaneously. these plates were incubated for h for target gene expression knockdown, followed by challenging one plate with aiv pseudovirions and the other plate with marv pseudovirions. virions were replaced with fresh medium h post infection and the plates were incubated for additional h. luciferase assays were then performed to quantify virus infection. screen data quality is crucial for subsequent data analysis and hit selection. quality control of the screen is commonly indicated by the z' factor [ ] which assesses the separation between measured signals of the positive and negative controls in an assay plate. in our screens, we included three controls: nontargeting sirna as the negative control and sirnas targeting luciferase or atp v c as the positive controls. the pseudovirion carries a luciferase reporter gene, and aiv and marv virus entry is dependent on the low-ph environment in the endosome/lysosome which is regulated by vacuolar atpase. so sirna targeting luciferase or atp v c (a component of vacuolar atpase) is expected to significantly reduce the final luciferase activity level. in addition, these two positive controls can serve as transfection efficiency controls, monitoring sirna transfection. eight control sirnas of each type were arranged on the column or of a -well assay plate, as shown in fig. a . figure b represents the normalized signal distributions of the samples and the controls. the normalized signal was obtained by dividing the measured signal in each well by the median luciferase signal of all sample wells in a plate. it is clear that both sirnas targeting luciferase and atp v c greatly reduced luciferase signal level with little variance, confirming their efficacy and excellent sirna transfection efficiency in the screen. the samples show a broad bell-shaped signal distribution, with a little positively skewed. the signal distribution of the non-targeting control was expected to overlap with that of samples. however in our screen, this distribution was shifted to the right, showing a higher signal level. the skewed sample distribution and the shift of negative control distribution suggest that the assay plates were affected by edge effects or the positional effects (see below). despite of the adverse impact of the positional effects, the z's calculated from two control combinations (luciferase versus non-targeting and atp v c versus non-targeting), are very close to . , an indication of excellent assay quality (fig. c ). in comparison with z's from aiv screen plates, z's from marv screen plates exhibited less favorable values and wider ranges, suggesting a larger the normalized signal distributions in both marburg virus (marv) and influenza h n virus (aiv) plates are plotted for sample (red) and controls: atp v c (green), luciferase (yellow) and non-targeting (blue). c z' factors. z' factors are computed either by the normalized signal values of luciferase and non-targeting controls (luc vs nt) or those of atp v c and non-targeting controls (atp vs nt). these z' factors are plotted for both marv and aiv plates variation in marv screen data. this is consistent with what is shown in fig. b , that is that both samples and non-targeting controls from marv screen plates have flatter signal distributions, as compared with those from aiv screen plates. in the aforementioned screen strategy, we were more interested in identifying virus-specific host proteins which could be revealed by a comparative analysis approach, due to the parallel nature of the screen. in this approach, the infection rates for aiv and marv for each sirna were obtained respectively by normalizing the measured luciferase signals with corresponding plate signal medians; the relative infection index, namely the logarithm of the ratio of their infection rates to base -log (ratio marv/aiv ), was then calculated for each sirna to indicate specificity. theoretically, an sirna with relative infection index equaling to zero means no bias of the sirna on the infections of both viruses; an sirna inhibiting marv infection or enhancing aiv infection results in a minus value, and an sirna with an the opposite effect results in a positive value. we used this comparative analysis as a means to correct the observed positional effects. the robust z-score, a robust version of z-score which indicates the number of standard deviations from the mean, was employed to represent data variance. the robust z-score distributions of the normalized signals for the columns and rows from both aiv and marv screen plates are shown in fig. . consistent with what we show in fig. b , the distributions from the columns and rows are skewed towards the high z-score value area, with the outer two columns and rows more severely affected. when the relative infection index was introduced, the excess variations of the peripheral positions were greatly reduced and the distributions of columns and rows were corrected to be more symmetrical. the same is true regarding the distributions of the all sirnas. as shown in fig. (the fig. comparative analysis reduces systematic positional effects. the normalized signals from marburg virus (marv) or influenza h n virus (aiv) plates are used to calculate the median and median absolute deviation (mad) respectively. relative infection index is first calculated by using normalized signals from both virus plates and then it is used to compute the median and the mad. the robust z-score for normalized signal or the relative infection index is then computed for each well and the robust z-score distributions across all the rows or the columns or the plates are plotted accordingly for aiv and marv plates bottom panel), the robust z-scores of the normalized signals from both screens are heavily skewed to the right, with almost no sirnas showing robust z-score less than − , a recommended criterion for picking inhibitory hits. the robust z-scores of the relative infection index, however, are evenly distributed, reducing the skewed distribution caused by the positional effects and making the data more suitable for the hit selection. since the relative infection index approach can reduce the systematic errors, we combined it with the individual virus infection rates to identify virus-specific host factors. as presented in fig. a , the hit selection began with normalizing the measured luciferase signals by the plate signal medians for marv and aiv, respectively. these normalized scores were used for calculating the relative infection index, from which a robust z-score was then computed for each sirna. thresholds of ± were adopted for filtering the hits. from the total , individual sirnas, , sirnas targeting , genes had robust z-scores less than − , while , sirnas targeting genes had robust z-scores greater than . another criterion ( sirnas per gene rule) that a gene must have at least sirnas showing similar effects was applied to further filter the hits, with the aim of reducing false positive numbers. accordingly, genes with sirnas for marv and genes with sirnas for aiv were selected, respectively. the individual virus infection rates (the normalized signals) were then employed as a reference to distinguish an inhibitory hit from the one that enhances virus infection. the robust z-scores are plotted against normalized signals of each virus in fig. b and c. a robust z-score less than − in fig. b means that the marv infection rate is much less than aiv infection rate for the corresponding sirna. this may be a result of inhibited marv infection or of enhanced aiv infection. thus a marv score (normalized signal) below . was used to indicate a marv inhibitory hit, which is represented in area i in fig. b . a gene with at least two plates is plotted against the robust z-score of the relative infection index. hits in area i are classified as specific inhibitory hits against either marv or aiv entry. hits in area ii are classified as non-specific inhibitory hits against either marv or aiv entry. hits in area iii are classified as hits that enhance virus entry of either marv or aiv sirnas' marv scores < . was used to filter the hits in area i, with genes being identified as host factors that inhibit marv entry. on the contrary, marv scores greater than . , which means the sirna has little effect on inhibiting marv infection, were used to indicate a hit enhancing aiv entry. area iii in fig. b represents these hits and host factors were identified with the sirnas per gene rule. similarly, area i in fig. c represents aiv inhibitory hits and genes were identified; area iii represents the hits enhancing marv entry with genes finally identified. the use of relative infection index together with individual virus infection rate and the sirnas per gene rule is a powerful means to identify virus-specific host factors and to reduce the false positive rate. the parallel nature of the screen strategy makes the results from the two viruses highly comparable and the relative infection index obtained by comparison easily establishes virusspecificity. as shown in fig. b and c, only hits in areas i and iii were picked for further analysis. though many other sirnas also showed inhibitory effects (area ii), these sirnas were ruled out for lack of virus-specificity, greatly reducing the false positive rate. further, the sirnas per gene rule helped to reduce false positive which may result from off-target effects or systematic errors. a selected short list of ebov or aiv entry-related genes is shown in table to demonstrate the power of the parallel screen strategy described in this report. four of seven subunits of the coatomer (cop-i) vesicular transport complex (i.e., arcn , copa, copb , and copg) were reported to be critical for influenza virus replication by others [ ] , but only arcn was identified as an aiv-specific hit in this screen ( table ). the other three proteins were identified in our screen as "shared" host factors for both aiv and marv (data not shown). proton-transporting v-type atpase was implicated as the host factor for influenza virus infection by the previous screens [ ] , and we show that only two subunits of atpase, apt v c and atp ap , are aiv-specific. a number of other subunits (i.e. atp ap , atp v b, atp v d , atp v a and atp v b ) are not specific to aiv but are "shared" host factors by both viruses. as for marv, a few essential marv-specific host factors were identified by the screen in this study. cathepsin l (ctsl), which primes the filovirus glycoprotein in the endosome [ ] , ext [ ] , which is involved in biosynthesis of heparan sulfate, and niemann-pick disease, type c (npc [ , ] ), showed strong bias against marv entry (table ) . also, hops complex, which mediates fusion of the endosome and lysosome, was identified as a host factor for ebola virus infection in a gene-trap based screen [ ] , and we identified vps , a subunit of hops complex, as a marv-specific hit in the current screen. it is interesting to note that two host proteins, folr [ ] and tim [ ] , which were previously reported as host factors important for filovirus entry, were not identified as the hits in this study ( table ). the results from this report are consistent with a study which showed that folr was not critical for filoviral entry [ ] . however, more studies are needed to evaluate the potential role of tim in filoviral entry. this report describes a new strategy, referred to as a parallel genome-wide rnai screen, to identify host factors which are important for entry of enveloped viruses. this strategy is particularly useful for highly pathogenic viruses such as ebola/marburg viruses and avian influenza virus h n (or bird flu) since the hiv- based surrogate system allows us to perform high throughput screens in the biosafety level (bsl- ) containment instead of the bsl- or bsl- for the infectious viruses with alleviated safety concerns. more importantly, the parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. the power of this strategy is well illustrated by a genome-wide rnai screen for identifying the host factors important for marv and/or aiv as described in this study. genome-wide rnai screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. however, it is clear that these rnai screens, even performed on the same virus by different groups, do not always identify the same set of host proteins, suggesting that high throughput rnai screening strategies need to be carefully developed and optimized prior to the actual screening. thus several features of the screening strategy described here are attractive to identify host proteins involved in viral entry of highly pathogenic enveloped viruses. one obvious advantage of this strategy is that it can distinguish virus-specific hits from the "shared" hits. because each virus in the screen also serves as a control for the other virus, an sirna showing effects on one virus but not on the other one will be tentatively classified as a specific hit, as illustrated in fig. . on the other hand, if an sirna shows similar inhibitory effects on both viruses, the scenarios are more complicated: ( ) the sirna may be toxic to the cells; ( ) it may induce an off-target effect; ( ) it may target an hiv- related process after the pseudovirion is released from the endosome/lysosome; ( ) it may target the shared host factors by both viruses (i.e. aiv and marv in this study). another merit of the current strategy is that it can reduce the false positive rate. the reasons for high false positive rates in a genome-wide rnai screen are due to a lack of replicates and sirna off-target effects. in this study, each sirna was actually tested twice, but with two different viruses. for an sirna specific to one virus entry, the possibility of obtaining similar results for two different viruses is pretty low, greatly decreasing the false positive rate. for shared hits, the false positive rate also decreases owing to the duplicate nature of this strategy. further, an sirna hit due to an off-target effect is likely to affect both viruses' infection and can thus be classified as a "shared" hit, resulting in a lower false positive rate for the virus-specific hit. in addition, it has been recently shown that virus infection rate is largely determined by the population context (e.g. local cell density) of the target cell which can be affected by gene perturbations through rnai [ ] . because the screens in this study were performed in parallel, the cell population context was, to a large extent, the same for the two viruses, making the results from the two screens highly comparable. we have observed the position effects in this study, which are a commonly observed phenomenon in rnai screens, as a result of the long incubation period needed for rnai assays. thus it can bring more noise to the screen and results in distortions of the true effects of sirnas. a number of pre and post-screening correction methods have been developed to battle these effects. it has been suggested that the controls evenly scattered over an assay plate by careful plate design can be used to adjust the systematic errors [ ] . however, most commercial available sirna libraries only have the peripheral wells reserved for controls; in our case, only the outer and columns are available for controls, leaving little room for control-based systematic error adjustment. also, a few mathematical algorithms have been proposed to reduce the effects [ ] . these methods usually assume a large difference between a positive and a negative response and the sparseness of positive responses, which is not true for rnai screens. in fact, rnai is a very complicated biological phenomenon; even an sirna targeting an unrelated gene may induce somewhat positive phenotype. thus modifying the original screen data with additional correction factors by those proposed mathematical treatment may lead to more man-made artifacts. in our comparative analysis approach, however, we are able to alleviate the positional effects without introducing a correction factor. the positional effects are largely due to the different evaporation rates across the plate which leads to a multiplicative bias to the measured signal. because the screens were performed in parallel, the duplicate wells with a same relative position in two plates were screened simultaneously, making them subject to a similar positional effect. this multiplicative bias can thus be corrected by simply dividing virus infection rate by the rate of the other one. in our case, the relative infection index was used to reduce the bias and the results we presented in fig. have clearly validated this approach. it should be pointed out that this report describes a powerful high throughput screening strategy which allows the initial identification of host proteins which may play a role in aiv-or marv-specific entry. however, the role of each putative host factor has to be individually confirmed and validated, and one of the challenges is to prioritize and select the host factors to be carefully characterized. we have developed a parallel genome-wide rnai screen strategy to identify specific host factors to either aiv or marv entry. implementation of this strategy generated two sets of data and a comparative analysis approach is proposed. our screening strategy together with the unique comparative analysis approach alleviates the systematic positional effects, makes the data more suitable for hit selection, and enables us to pick virus-specific hits with a much lower false positive rate. this strategy, we believe, can be easily adapted to other screens with the aim of increasing screen specificity and reducing false positive rates. human t embryonic kidney cells and human lung epithelial a cells were cultured in dulbecco's modified eagle's medium (dmem, cellgro) supplemented with % fetal bovine serum (fbs, gibco), μg/ml of streptomycin and units of penicillin (invitrogen). the avian influenza virus h n /hiv pseudovirions (aiv) and marburg virus/hiv pseudovirions (marv) were generated from the following plasmids: hemagglutinin (ha), isolated from a highly pathogenic avian influenza virus, a/ goose/qinghai/ / (h n ) [ ] ; neuraminidase (na) from a/puerto rico/ / (h n ) [ ] ; marburg virus glycoprotein gp (mgp) [ ] . the hiv- proviral vector pnl - .luc.r − e − [ , ] was obtained through the nih aids research and reference reagent program. aiv and marv pseudovirions were produced by transient co-transfection of human t cells using a polyethylenimine (pei)-based transfection protocol [ ] . replicationdefective hiv vector (pnl - .luc.r − e − ) together with plasmids encoding mgp or ha/na were used for transient co-transfection into t producer cells. six hours after transfection, cells were washed with phosphatebuffered saline (pbs), and ml of fresh medium was added to each plate ( mm). forty-eight hours post transfection, the supernatants were collected and filtered through . μm pore size filter (nalgene). the pseudovirion stocks were stored at °c prior to use. three sirna libraries (silencer® select human druggable genome sirna library v , human druggable genome v extension and human genome v sirna extension) were purchased from ambion (austin, tx). these libraries contain , sirnas targeting , human genes (each gene has three distinct sirnas). daughter plates were prepared in -well format at a nm concentration in water. controls include sirna targeting atp v c or luciferase and non-targeting sirna. all control sirnas were purchased from ambion. all sirnas were arrayed in -well plates. for each assay plate well, μl sirna was mixed with . μl lipofectamine rnaimax (invitrogen) and . μl opti-mem (invitrogen). after min incubation at room temperature, μl cell suspension of a cells was added, resulting in a final sirna concentration of nm. cells were incubated at °c and % co for h. then the medium was removed and the cells were challenged with μl marv or aiv virions in parallel. virions were removed h later and μl fresh dmem supplemented with fbs and antibiotics was added to each well. after another h incubation, μl of neolite luciferase substrate (perkinelmer, waltham, ma) was mixed in, incubated for min, and luciferase activity was measured with an envision plate reader (perkinelmer). all multi-well pipetting procedures were conducted by the janus automated liquid handling system (perkinelmer). the measured luminescence signal was analyzed using the statistical programming language r. the signal in each well was first normalized by the median value of all the samples from the same plate. the z' factor [ ] was calculated from the normalized signals of not-targeting and atp v c controls or from those of not-targeting and luciferase controls on each plate with the following equation: z ' = − (std highsignal + std lowsignal )/(mean highsignal − mean lowsignal ). for each sirna, the relative infection index was calculated from the corresponding normalized signals from the marv and aiv plates with the following equation: relative infection index = log (normalized signal marv / normalized signal aiv ). robust z-score was calculated for both normalized signal and relative infection index. first, median absolute deviation (mad [ ] ) was calculated as follows: mad ¼ : Â median x ij −median x ð Þ À Á ; and then the robust z-score was calculated for each sirna as follows: where x represents the set of all the normalized signals or the relative infection indexes of the samples and x ij indicates the value for a particular well at row i and column j. the constant . is used to make mad comparable to standard deviation. 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receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection single-cell analysis of population context advances rnai screening at multiple levels integrating experimental and analytic approaches to improve data quality in genome-wide rnai screens quantitative assessment of hit detection and confirmation in single and duplicate high-throughput screenings human immunodeficiency virus type viral protein r (vpr) arrests cells in the g phase of the cell cycle by inhibiting p cdc activity vpr is required for efficient replication of human immunodeficiency virus type- in mononuclear phagocytes submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this research was partially supported by national institutes of health (usa) grant ai to l. r. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.authors' contributions hc, kk, ao, mw and eb participated in study design and performed the sirna screen. hc conducted data analysis. lr conceived the study, participated in its design and coordination. hc and lr wrote the manuscript. ev critically reviewed the manuscript. all authors read and approved the final manuscript. key: cord- - vim wno authors: zandi, keivan; teoh, boon-teong; sam, sing-sin; wong, pooi-fong; mustafa, mohd rais; abubakar, sazaly title: antiviral activity of four types of bioflavonoid against dengue virus type- date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vim wno background: dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. in the present study, antiviral activity of four types of bioflavonoid against dengue virus type - (denv- ) in vero cell was evaluated. anti-dengue activity of these compounds was determined at different stages of denv- infection and replication cycle. denv replication was measured by foci forming unit reduction assay (ffura) and quantitative rt-pcr. selectivity index value (si) was determined as the ratio of cytotoxic concentration (cc( )) to inhibitory concentration (ic( )) for each compound. results: the half maximal inhibitory concentration (ic( )) of quercetin against dengue virus was . μg ml(- )when it was used after virus adsorption to the cells. the ic( )decreased to . μg ml(- )when the cells were treated continuously for h before virus infection and up to days post-infection. the si values for quercetin were . and . μg ml(- ), respectively, the highest compared to all bioflavonoids studied. naringin only exhibited anti-adsorption effects against denv- with ic( )= . μg ml(- )and its related si was . . daidzein showed a weak anti-dengue activity with ic( )= . μg ml(- )when the denv- infected cells were treated after virus adsorption. the si value for this compound was . . hesperetin did not exhibit any antiviral activity against denv- . the findings obtained from foci forming unit reduction assay (ffura) were corroborated by findings of the qrt-pcr assays. quercetin and daidzein ( μg ml(- )) reduced denv- rna levels by % and %, respectively. there was no significant inhibition of denv- rna levels with naringin and hesperetin. conclusion: results from the study suggest that only quercetin demonstrated significant anti-denv- inhibitory activities. other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of denv- virus replication. these findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. this group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication. dengue virus (denv) is a member of the genus flavivirus of the flaviviridae family. it is a significant human pathogen which causes a wide spectrum of clinical illnesses ranging from a silent or mild febrile infection, self-limited dengue fever (df) to the severe dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). there are four dengue virus genotypes, denv- , denv- , denv- and denv- which are transmitted to humans mainly by two species of mosquitoes, aedes agypti and aedes albopictus [ ] . all four denv can cause dengue. to date there are no effective vaccine or antiviral treatment for dengue. dengue patients are usually supportively-treated until they recover without any specific treatment measures. several studies have shown that the level of viremia correlates with the severity of disease with high viremia often seen in severe dengue. hence, antivirals that can reduce the level of viremia or the viremic phase could possibly reduce the severity of dengue. plants and plant's derived compounds remain an important source for the discovery and the development of new antiviral drugs because of their expected low side effects and their high accessibility in the nature [ ] [ ] [ ] . there have been numerous reports on the antiviral activity of various phytochemicals against dengue viruses and these include various flavonoids [ ] [ ] [ ] [ ] . flavonoids are basically low molecular weight phenolic compounds found widely in different kinds of plants. different types of flavonoids can be found in fruits, roots, nuts, seeds, bark, steams and flowers of plants. these include quercetin which can be found in some foods and fruits such as green and black tea, apple, onion, citrus, tomato and some other plants [ , ] . antiviral activities of various other flavonoids have also been reported against some viruses including human cytomegalovirus (hcmv), hsv- , hsv- and some types of human adenoviruses [ ] [ ] [ ] . in the present study, we are interested to examine the anti-dengue virus properties of quercetin, hesperetin, naringin and daidzein. hesperetin is a flavonone and its glycoside form, hesperidin is water soluble and it could be found in various citrus fruits. after ingestion of hesperidin, its sugar moiety is released from the backbone of this compound and the aglycone form known as hesperetin can be released. in vitro antiviral activities of hesperetin have been reported against some rna viruses [ ] [ ] [ ] . naringin on the other hand, is a flavonone glycoside found abundantly in grapefruit juice. antiviral activity of naringin were reported against hsv- and hsv- but this finding remains controversial [ , ] . daidzein is an isoflavone found in soybeans and its antiviral activity against influenza viruses has been reported [ ] . currently, there is no published data on the possible anti-dengue virus activities of quercetin, hesperetin, naringin and daidzein. therefore, in this study we evaluated these compounds activities on denv- (ngc strain) replication in cell culture system. the effects of each compound were evaluated against different stages of dengue virus replication including virus adsorption, intracellular replication and direct virucidal activities. four different types of bioflavonoid, quercetin, naringin, hesperetin (sigma-aldrich, st. louis, mo, usa) and daidzein (indofine chemical co. inc., hillsborough, nj, usa) were evaluated for their potential activities against dengue virus replication. dimethyl sulfoxide (dmso) (sigma-aldrich, st. louis, mo, usa) was used to dissolve the lyophilized form of compounds and prepared stock solutions ( mg ml - ) were stored at - c. stock solution was diluted using cell culture medium and sterilized by a syringe filter with . μm pore size (millipore, ma, usa) right before each experiment. c / mosquito cell line derived from aedes albopictus and vero (african green monkey kidney) cell line were used in this study. both cell lines were maintained and propagated in eagle's minimum essential medium (emem) (gibco, ny, usa) containing % fetal bovine serum (gibco, ny, usa). cultured c / and vero cells were incubated at c and c, respectively in % co humidified chamber. at the time of virus propagation, serum concentration was reduced to %. dengue virus type- (denv- ) new guinea c strain (ngc) was propagated using c / cell line and harvested after cpe presentation on day seven post-infection. after titration, viral stock was stored at - c. cell lines and virus were provided by virology laboratory of the tropical infectious disease research and education center, faculty of medicine, university of malaya (kuala lumpur, malaysia). mtt assay was performed following the manufacturer's instructions (promega, wi, usa). briefly, confluent vero cells in -well cell culture microplates were treated with different concentrations of each compound in triplicate. the treated cells were incubated for four days at c followed by the addition of μl of mtt solution to each well. the microplate was incubated at c for h. then, μl of the solubilisation/stopping solution was added to each well. the optical density (od) of wells was measured at using -well plate reader (tecan, mannendorf, switzerland). dose-response curves were plotted using graph pad prism (graph pad software inc., san diego, ca). in order to determine the prophylactic anti-dengue activity of compounds, different concentrations of compounds were added to the vero monolayer cells in triplicate at different times, h before virus infection. after h of pre-infection treatment, the cells were washed twice with sterile pbs and then ffu of denv- was inoculated to the cells and incubated at c for h. to determine the effects of continuous treatment, different concentrations of each compound were added to the vero cells, h pre-infection and continuously for days post-infection. in a separate experiment, antiviral activity of compounds against intracellular replication of denv- was performed by inoculation of ffu of virus to each well in triplicate. after adsorption of virus to the cells for h at c, the cells were washed with pbs to eliminate the unabsorbed viruses. then, different concentrations of each compound were added to the cells, followed by days of incubation at c. denv rna was then quantified using quantitative rt-pcr. in another experiment, vero cells at % confluency were infected with ffu of denv- in the presence or absence of different concentrations of each compound. the microplate was kept at c for h for virus adsorption. then the cells were washed two times by sterile pbs and incubated at c for four days. direct virucidal effects of the bioflavonoids were investigated by incubating denv- suspension containing ffu with an equal volume of the different concentrations of each compound for h at c. then, vero cells were infected with the treated viral suspension in triplicate. after h adsorption at c, cells were washed twice with pbs in order to remove unabsorbed viruses. then the microplate was incubated at c for days. antiviral activities of the tested compounds were evaluated by measuring the reduction in number of viral foci. briefly, confluent monolayers of vero cells were prepared in wells cell culture microplate. then the infected cells treated using different procedures described above were overlaid with . % of carboxy methyl cellulose (cmc) (sigma-aldrich, st. louis, mo, usa) containing emem. viral foci were visualized using peroxidase-based foci staining assay four days post infection [ ] . the numbers of denv- foci were counted using a stereomicroscope and the titer of virus was expressed as foci-forming-unit (ffu). the percentage of viral foci reduction (rf %) compared with controls was calculated as follows: rf (%) = (c-t) × /c. where, c is the mean of the number of foci for negative control well (without compound) and t is the mean of the number of foci in treated wells. reduction in the number of viral foci was further verified using quantitative rt-pcr (qrt-pcr). quantitative rt-pcr was performed to determine the effects of bioflavonoids on denv replication by quantifying denv- genomic rna copies based on a method described previously with some modifications [ ] . briefly, intracellular and extracellular denv- rnas were harvested from the denv-infected vero cells. viral rna was extracted using two types of rna extraction kits (qiaamp viral rna mini kit and rneasy mini kit) (qiagen, hilden, germany). quantitative rt-pcr assay was performed by adding μl of extracted denv rna to the sensimix sybr green reagent (quantace, watford, united kingdom) which contained . μl ddh o, μl x sensimix one-step, . μl x sybr green solution, units of rnase inhibitor, pmol of forward (dnf) and also reverse (d r) primers [ ] . all samples were assayed in triplicate. the amplifications were performed using the dna engine opticon system (mj research/bio-rad, hercules, ca) with the following thermal conditions: reverse transcription at °c for min, initial denaturation at °c for min, followed by cycles of °c for sec, °c for sec and °c for sec. melting curve analysis was subsequently performed at temperature from °c to °c to verify the assay specificity. for absolute quantitation of the viral rna, a standard curve was established with a serially diluted rna extracted from denv- stock with known titer. graph pad prism for windows, version (graph pad software inc., san diego, ca, ) was used to determine the cytotoxic concentration (cc ) and inhibitory concentration (ic ) values of bioflavonoids. selectivity index value (si) was determined as the ratio of cc to ic for each compound. mtt assay was used to determine cytotoxicity of each bioflavonoid on vero cells and the cc value of each compound was calculated (table and figure ). vero cells were treated by bioflavonoids for days which was the same duration used for antiviral activity assay. results showed that hesperetin with cc = . ± . μg ml - is the most cytotoxic compound for vero cells compared to the other tested compounds. quercetin and daidzein showed lower toxicity against vero cells at cc . ± . and . ± . μg ml - , respectively. meanwhile, naringin with cc = . ± . μg ml - showed the least cytotoxic effects against vero cells. cells treated with vehicle control, % dmso did not show any cytotoxicity against vero cells. results of vero cells pre-treatment with the compounds showed that μg ml - of quercetin could decrease the number of denv- foci by % ± . when compared to the non-treated cells. however, there was no prophylactic activity against denv- from other compounds (data not shown). in post-adsorption assay, quercetin exhibited the most significant antiviral activity against denv- amongst the bioflavonoids tested with ic = . μg ml - (figure a) . the si value for quercetin in post-adsorption assay was relatively high at . . it was also demonstrated that the level of denv- specific rna production in the presence of μg ml - of quercetin decreased by more than % ± when compared to the non-treated infected cells (figure b ). daidzein showed very weak anti-dengue activity with ic = . μg ml - when the infected cells were treated after denv- adsorption (figure a ). its related si value was . . the levels of denv- rna production in the presence of μg ml - of daidzein decreased by only . % ± . when compared to the non-treated infected cells (figure b ). naringin and hesperetin did not exhibit any anti-dengue activity when they were used after adsorption of denv- to the vero cells ( figure ). although there was no significant direct virucidal activity against denv- by quercetin, continuous treatment of cells from h before virus infection up to days post-infection exhibited anti-dengue activity with ic = . μg ml - (figure a) . the si value for continuous treatment with quercetin was . and higher than the si value ( . ) for post-adsorption assay. in addition, the level denv- rna production decreased by more than . % ± . when vero cells were treated with μg ml - of quercetin, h before virus infection and up to days post infection ( figure b ). there was no significant change in the antiviral activity of daidzein when cells were treated continuously from h before virus infection up to days post infection comparing to its anti-dengue activity for postadsorption treatment (figure ). no significant antiviral activity for naringin and hesperetin was observed for the continuous treatment against denv- ( figure ). however, naringin exhibited anti-adsorption activity when it was added to the cells at the same time of virus adsorption. the ic value for naringin was . μg ml - and its related si was . ( figure a ). there was a reduction of . % ± . in denv- rna production in the presence of μg ml - of naringin (figure a) . the majority of the viral foci in cells treated with μg/ml quercetin appeared smaller, less intensely stained and more diffused within the focus (figure b) , compared to the larger, well-defined and more intensely stained foci of the untreated cells (figure a ). this observation is consistent with the reduction of the percentage of foci and rna copy number. results from the direct virucidal activity evaluation of each compound showed that there was no extracellular inhibitory activity against denv- for all the tested figure anti-adsorption activity of flavonoids against denv- . flavonoids were added directly to virus inocula for h at c. the inocula were used to infect vero cell monolayers in wells cell culture microplates. the reduction in foci forming unit was calculated relative to the controls maintained in parallel (a) and the respective denv- rna copy numbers were quantified using qrt-pcr (b). data from triplicate experiments were plotted using graph pad prism version (graph pad software inc., san diego, ca). the reduction of intracellular replication of dengue virus by . % and % following treatment with μm of glabranine and -o-methyle glabranine, respectively [ ] . similarly, other synthetic flavonoid derivatives also showed antiviral activity in hepg cells [ ] . whereas, pinostrobin was reported to inhibit denv- ns b/ns protease an enzyme important in dengue virus replication in an in vitro study [ ] . these suggest that flavonoids as a group could consist of select compounds that possesses inhibitory activities against denv. to investigate which of the many flavonoids could affect denv infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of vero cells. unlike the previous studies which evaluated antiviral activity of flavonoids only after adsorption of virus to the cells [ , ] , the present study evaluated antiviral activity by different treatment procedures tailored to determine prophylactic, post adsorption, continuous treatment and direct virucidal activities of quercetin, naringin, daidzein and hesperetin. our findings demonstrated that quercetin was the only compound among all tested flavonoids that consistently showed significant antiviral activity against denv- in vero cells. selectivity indices for quercetin when the infected cells were treated or when uninfected cells were treated continuously h before infection until days post-infection were . and . , respectively. the noted differences between si values for quercetin could be due to the intracellular accumulation of quercetin during continuous treatment. a weak effect for prophylactic activity of quercetin however, was also observed. these findings suggest that the main anti-dengue activity of quercetin is likely due to its activity against the different stages of intracellular replication of denv- instead of early stages of its replication cycle such as virus attachment or entry. although, no direct virucidal activity or anti-attachment activity of quercetin was observed in the present study, antiviral activity of quercetin against human cytomegalovirus was reported with ic = . μm [ ] . quercetin was also reported to be effective against herpes viruses where it is more specific against hsv- with si = compared to hsv- with si = . [ ] . the mechanisms of how quercetin exerts its antiviral effects are not known. however, the effects of other flavonoids against cellular rna polymerases and formation of the complex with rna have been reported suggesting that quercetin could also affect the similar replication enzymes [ , ] . sylimarin, a flavonoid found effective against hepatitis c virus (hcv), another member of flaviviridae family [ ] , inhibits virus replication by inhibiting the activity of viral rna polymerase. in our study, results from the qrt-pcr supported the findings from the viral foci reduction assays that quercetin inhibits denv- replication and the significant reduction in the denv specific rna suggests that quercetin may also target the virus replication machinery, namely by inhibiting the rna polymerase. antiviral activity of naringin has been evaluated against few herpesviruses and rotavirus but their reported antiviral activities against hsv- and hsv- are inconclusive [ , ] . in addition it was reported that naringin did not exhibit any antiviral activity against another rna virus, sindbis virus [ ] . in the present study, the only anti-dengue activity of this flavonoid was demonstrated against adsorption and attachment of virus to the vero cells and based on its antiviral activity (ic = . μg ml - ) and its related selectivity index (si = . ), it may not be a good candidate for further development as anti-dengue drug. similarly, daidzein activity against denv- was not significant compared to quercetin (si = . ). continuous treatment of the infected vero cells from h before virus infection up to days post infection did not improve its anti-dengue activity significantly. this compound therefore, could not be a suitable candidate for further development as anti-dengue drug. hesperetin, the other flavonoid evaluated in our study, did not show no anti-dengue activity in any stages of virus infection and replication processes and this is despite the previously reported antiviral activity of hesperetin against sindbis virus [ ] . therefore, hesperetin is also not recommended for further investigations for anti-dengue drug development. in all our experiments, we showed that . % of dmso, the highest concentration of solvent used in the bioflavonoid treatment did not exhibit any antiviral activity against denv- and this eliminated any probable antiviral activity from dmso. findings from our study, suggest that there are select flavonoids including quercetin and fisetin, which are both flavonol, that exhibited significant denv replication inhibition properties [ ] . while the flavonoids in general share common basic molecular base structure, flavone ( -phenyl- , -benzopyrone), we showed here that the flavanone, hesperetin, and flavanone glycoside, naringin, showed no significant anti-denv replication activities. in addition, we had earlier shown that naringenin [ ] , another flavanone metabolized from naringin and here, daidzein, an isoflavone also had no significant denv replication inhibition properties. while quercetin was shown here to be effective in inhibiting denv replication, its glycoside form, rutin (quercetin- -o-rutinoside) showed no significant inhibition properties [ ] . these suggest that while flavonol could be the basic molecule that possesses anti denv replication properties, specific structural properties of the different flavonol derivatives would have different effects on the efficacy of the compounds against dengue. the demonstration in vitro that flavonols including quercetin and fisetin possess anti denv replication properties does not necessarily translate into immediate use of these compounds as antivirals against denv. further studies will be needed to demonstrate the antiviral activities of these compounds against different genotypes of dengue virus and in appropriate animal model. there is also a need to address the issue of the low bioavailability of quercetin especially for therapeutic use [ ] [ ] [ ] . several strategies to increase the bioavailability of quercetin that include using lipids and emulsifiers, co-crystalization of quercetin or using ester-based precursors have been investigated [ ] [ ] [ ] . the other topic of research would be combination drug study. at its current calculated ic values, the antiviral efficacy of quercetin can be further improved possibly by combining it with other potential anti-dengue compounds. this is exemplified in a study that reported the synergistic effect of αglucoside in combination with a standard antiviral drug, ribavirin is effective against dengue infection [ ] . in conclusion, the present study demonstrates that the bioflavonoid quercetin exhibited significant anti denv replication properties. we further showed that quercetin affects intracellular denv virus replication but not the denv attachment and entry processes. these results together with the earlier findings reporting the anti denv properties of fisetin, suggest that these 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quercetin in rats cocrystals of quercetin with improved solubility and oral bioavailability ester-based precursors to increase the bioavailability of quercetin combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo antiviral activity of four types of bioflavonoid against dengue virus type- we thank the ministry of science, technology authors' contributions kz designed and carried out the antiviral and cytotoxicity studies and drafted the manuscript. btt carried out the virus propagation and antiviral studies. sss participated in the quantitative rt-pcr. wpf participated in the design of the study, performed statistical analyses and edited the manuscript. mrm participated in study design and provided all bioflavonoids. sab conceived the whole study and edited the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -ik w t e authors: ali, s asad; gern, james e; hartert, tina v; edwards, kathryn m; griffin, marie r; miller, e kathryn; gebretsadik, tebeb; pappas, tressa; lee, wai- ming; williams, john v title: real-world comparison of two molecular methods for detection of respiratory viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ik w t e background: molecular polymerase chain reaction (pcr) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (dfa) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. we sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories. methods: we tested nasal and throat swab specimens obtained from infants with respiratory illness for common respiratory viruses using both a multiplex assay (respiratory multicode-plx assay [rma]) and individual real-time rt-pcr (rt-rtpcr). results: both assays detected viruses in more than % of specimens, but there was discordance. the rma assay detected significantly more human metapneumovirus (hmpv) and respiratory syncytial virus (rsv), while rt-rtpcr detected significantly more influenza a. we speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza a in the rma assay, and for hmpv and rsv in the rt-rtpcr assay. the tests were then repeated and again compared. the new primers led to improved detection of hmpv and rsv by rt-rtpcr assay, but the rma assay remained similar in terms of influenza detection. conclusions: given the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other pcr based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy. respiratory viruses are common causes of human disease. molecular detection techniques have allowed previously known viruses to be more reliably identified and new viruses to be discovered [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . molecular techniques such as real-time rt-pcr (rt-rtpcr) can be performed for each individual virus, or they can be combined into a multiplex rt-rtpcr assay. other molecular methods that can target a single analyte or multiple viruses use a variety of nucleic acid detection based strategies, including microarrays [ ] , mass spectrometry [ ] , spectrally distinct bead microarrays [ ] , capillary electrophoresis [ ] , or microsphere flow cytometry [ ] . the performance of molecular diagnostic methods, either rt-rtpcr or multiplex assays, typically has been evaluated by comparing them with conventional direct fluorescent antibody (dfa) or culture, with few direct comparisons reported between molecular assays. moreover, there are few reports of laboratories collaboratively comparing methods between institutions. in this study, we compared a multiplex respiratory virus panel (respiratory multicode assay [rma], eragen biosciences and u. wisconsin-madison) with rt-rtpcr (vanderbilt university) for the detection of common respiratory viruses. nasal and throat swabs were collected from children ≤ months old who were admitted to the monroe carell jr. children's hospital at vanderbilt from september , through may , with acute respiratory illness, as part of the tennessee children's respiratory initiative [ ] . all specimens collected during this period were included in the study. within four hours of collection, the samples were divided into aliquots, lysis buffer added, and specimens were frozen at - °c. separate unthawed aliquots were sent to the research laboratories at vanderbilt university and university of wisconsin-madison for rt-rtpcr and rma testing, respectively. single rt-rtpcr experiments were performed for the following viruses: respiratory syncytial virus (rsv), influenza a and b, human metapneumovirus (hmpv), human rhinovirus (hrv), parainfluenza , and (piv , piv , piv ), and human coronaviruses netherlands (nl- ), oc , and e. rna was extracted on an automated instrument (magnapure total nucleic acid extraction kit; roche applied science), and refrozen at - °c until each individual rt-rtpcr was performed. primers and probes for hcov-nl , oc , e, rsv, hmpv and hrv were obtained from the published literature [ ] [ ] [ ] [ ] [ ] [ ] . influenza primer and probe sequences were provided by steve lindstrom, centers for disease control and prevention [ ] . primers and probes for piv , and were designed using primer express version . (applied biosystems). primer and probe sequences, concentration, and annealing temperatures are listed in table . twenty-five-μl reaction mixtures containing μl of specimen rna were tested using the quantitect probe rt-pcr kit (qiagen) on a smart cycler ii (cepheid). cycling conditions were min at °c, min at °c, and cycles of seconds at °c and seconds at - °c (table ). all rt-rtpcr assays were optimized and characterized using rna runoff transcripts and were capable of detecting < rna transcript copies/reaction (data not shown). all samples were tested by a commercial rt-rtpcr assay for human beta-actin mrna (applied biosystems) to ensure rna integrity. rma is a multitarget, high throughput technology that integrates multiplex pcr and microsphere flow cytometry [ , , ] . while the luminex technology can detect up to targets in one reaction, this version of the assay included viral targets. we report results for the same viruses tested by the rt-rtpcr assays. nucleic acid was extracted with trizol reagents. the rma assay was performed with primers and conditions as previously described, including the addition of an external insect virus spiked into each specimen to control for rna extraction, rt, and pcr [ ] . both sites were blinded to the results from the other laboratory until all data were analyzed. in order to address potential weaknesses indicated by discordant results between the two assays in the first round of comparison, both rt-rtpcr and rma testing were modified for a second round of testing. the first round primers and probes for rsv and hmpv [ , ] were changed for the rt-rtpcr assay [ ] and (klemenc et al, unpublished data) and all samples were retested for these two viruses. in the rma assay, the primers and probe for influenza a were modified and the cdna synthesis protocol was changed from promega reagents to the applied biosystems high-capacity cdna kit. demographic and clinical variables of the study sample are described as frequencies and percent or median and interquartile ranges [iqr] as appropriate. the rma and rt-rtpcr assays were compared using the test for inter-rater agreement (kappa coefficient). kappa coefficient was calculated for any comparison in which the value of all data cells was ≥ , with a kappa ≥ . considered to indicate good agreement. bootstrapping was used to estimate % confidence limits of kappa. cycle threshold (ct) numbers for influenza and rsv were compared between groups of specimens using the t-test. a total of infants hospitalized with respiratory symptoms were enrolled from september through may . samples from children were available for study in both sites; three specimens were only available for rma testing. study children were % male with a median age of weeks [iqr - weeks]. the admitting diagnoses of these infants were bronchiolitis ( %), upper respiratory infection ( %), and other ( %). rates of viral detection were compared using both assay methods. restricting the analysis to the viruses tested with both methods, at least one virus was detected in ( . %) samples by rma and in ( . %) samples by rt-rtpcr. a total of and viruses were identified in these samples by rma and rt-rtpcr, respectively (table ) . in the first round of testing, rma detected more rsv (n = , %) than rt-rtpcr (n = , %). the kappa coefficient between the two tests was . . rma also differentiated between rsv sub-types a and b. of the rsv positive samples detected by rma, were rsv a and were rsv b (one sample had both rsv a and rsv b). of the rsv a identified through rma, rt-rtpcr detected rsv in ( %) of them. however, of the rsv b detected by rma, rt-rtpcr only detected rsv b in ( . %) samples. in the second round, the primers and probes for the rsv rt-rtpcr assay were redesigned based on further analysis of published viral sequences [ ] . using these new primers and probes, rsv was identified in ( %) samples with rt-rtpcr. the inter rater agreement for rsv between the repeat rt-rtpcr and the first rma was . . of the rsv a and rsv b detected by the rma assay, the repeat sample analysis detected rsv in and respectively. samples were positive for rsv with repeat rt-rtpcr testing but negative for rsv with rma testing; the mean ct for these specimens was higher than the mean ct for specimens detected by both assays ( . ± . vs. . ± . , p < . ). in the first round, the rt-rtpcr detected influenza a in ( . %) samples while the rma detected influenza a in ( . %) samples. rt-rtpcr detected influenza b in sample while rma detected influenza b in a different sample. in the second round of testing, the influenza a rma primers and probes were redesigned. compared to samples positive for influenza a by rrt-pcr, the repeat rma assay detected positive samples; all were also detected by the rt-rtpcr assay. the mean ct of the influenza a specimens detected by the rma assay was lower than the mean ct of all influenza a specimens ( . ± . vs. . ± . , p < . ). rt-rtpcr detected hrv in ( . %) samples as compared to the rma, which detected hrv in ( . %) samples (kappa coefficient . ). in two of the discordant samples, rt-rtpcr detected hrv while the rma detected enterovirus. during the first round of testing, rma detected hmpv in ( . %) samples while rt-rtpcr detected hmpv in ( . %) samples. in the second round using redesigned rt-rtpcr hmpv primers and probes, hmpv was detected in samples, of which were concordant with the results of the first rma assay. the first rma assay detected hmpv in additional samples that were not detected by the repeat rt-rtpcr assay, while one sample positive for hmpv in the first rma was negative in the second set of tests. nl was detected in ( . %) samples through rma and in ( . %) samples through rt-rtpcr. hcov oc was detected in ( . %) samples through rma and in ( . %) samples through rt-rtpcr. hcov e was not detected in any sample by either assay. piv was detected in ( . %) samples through rma and in ( . %) samples through rt-rtpcr. piv was detected in ( . %) sample through rma and in ( . %) sample through rt-rtpcr. no piv was detected by either method. the comparison of the first and second run of rma assay is summarized in table . the most notable discrepancies between runs were for hrv and rsv. the sensitivity and specificity of multiplex viral diagnostic assays have generally been compared to viral culture and dfa [ ] , instead of more sensitive rt-rtpcr methods [ , [ ] [ ] [ ] . comparison of molecular methods with other sensitive molecular techniques, such as rma with rt-rtpcr, is appropriate to determine whether the sensitivity for each analyte is sufficient for clinical or research use. although both assays performed well for selected viruses in this study, the first round of comparison revealed differences in the rates of detection for specific viruses ( table ). the rma assay detected significantly more hmpv and rsv, while the rt-rtpcr assay detected more influenza a and hrv. redesigning primers led to substantial improvements in the detection of hmpv and rsv by rrt-pcr, while there was little improvement in the performance of rma for influenza virus with the change in primers. this discordance could have been due to sequence variation; there was insufficient residual specimen to sequence the viruses. however, both influenza a assays targeted regions of the matrix gene that are highly conserved among genbank sequences (data not shown). the mean ct of the influenza and rsv specimens detected only by the rt-rtpcr assay was significantly higher than those detected by the rma assay, suggesting that the discordance may have been due to the sensitivity of the two assays for specimens with a lower viral load. one recent study compared a commercial version of the rma known as plx-respiratory viral assay (plx-rvp) to rt-rtpcr and found good concordance, but only tested a portion of the plx-rvp negative samples [ ] . the rma and plx-rvp differ both in primer sequences, and in the method used to extract rna and perform reverse transcription. both of these factors are important determinants of assay sensitivity. given that rna viruses are prone to mutation [ ] , currently circulating field strains of these viruses should be regularly monitored for sequence divergences that would affect primers and probes used in pcr-based assays. this is particularly true for influenza, which undergoes considerable variation every year. molecular assays are often targeted to conserved internal genes, but this is based on known sequences. further, due to segment reassortment and antigenic shift, new internal gene segments may arise among influenza viruses infecting humans. for multiplex assays such as the rma, inclusion of two probe sets that target two different parts of the viral genome might eliminate the risk that a single mutation would lead to nondetection. our study has some limitations. first, we did not use identical extraction methods and primers in the rt-rtpcr and rma assays, and therefore discrepant results could have been due to differences in these processes or primer design. however, this was an intentional choice, since the specific goal of this study was to compare two "real-world" methods. both laboratories have published extensively in the area of respiratory virus diagnostics and epidemiology, and have evaluated their independent methods. this study shows that collaboration between groups offers a valuable tool to assess assay performance impartially. second, we did not use a gold standard test to confirm presence or absence of virus in cases of discrepancy between the two assays. we explored the option of sequencing of the discordant samples to detect sequence differences or false positives, but there was not enough residual specimen to allow this. nonetheless, we repeated the testing of viruses that had major discrepancies with the assumption that if the repeats were positive, it provided reassurance that the positive samples were true positives. third, the rt-rtpcr was only repeated for hmpv and rsv due to limited sample; there could have been discordances for other viruses. fourth, the rma assay was repeated with altered influenza primers while other primers were unchanged; it is possible that a change in influenza primers had an impact on the overall performance of the assay. one important consideration for any multiplex assay is the need to re-optimize for all targets when a change is made in any of the primers used. finally, the results were not evaluated by dfa or culture, though the objective was to directly compare sequence-based methods. in summary, for most viruses tested, there was good agreement between the two assays. however, discrepancies highlight the need for similar comparisons between molecular assays on a routine basis, so that weaknesses can be promptly identified and corrected. while investigators may be reluctant to make comparisons that might suggest deficiencies of particular methods, collaborative studies such as ours are critical to advance these diagnostic methodologies. two other recent studies comparing molecular methods identified similar discrepancies [ , ] . in addition to clinical specimens validated by culture, respiratory virus proficiency panels distributed by quality control programs like the european quality control for molecular diagnostics (qcmd) [ ] , cdc, or college of american pathologists, or commercial proficiency panels (e.g., zeptometrix nattrol™ respiratory validation panels) provide an external quality control. clearly, the overall comparison between molecular methods should consider the performance of each individual analyte. the goal of this study was not to determine which is the "best" assay; the choice of assay for a given purpose depends on many factors, including cost, available equipment and expertise, sample volume, and others. multiplex assays may offer substantial savings in time and cost due to multiple analytes, and are generally preferred for clinical testing. comparison of molecular diagnostic methods to culture and dfa is useful to establish validity, and ensure that cultivable viruses are correctly identified by sequence-based technology. however, our data show that molecular diagnostic methods also must be compared to other molecular techniques to assess performance, particularly because of the superior sensitivity of the molecular methods compared to culture and dfa. detection of respiratory viruses by molecular methods superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children clinical evaluation of nuclisens magnetic extraction and nuclisens analyte-specific reagents for real-time detection of human metapneumovirus in pediatric respiratory specimens human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human rhinoviruses: the cold wars resume identification of a new human coronavirus osterhaus ad: a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples microarray-based detection and genotyping of viral pathogens diagnostic system for rapid and sensitive differential detection of pathogens principles of the xtag respiratory viral panel assay (rvp assay) comparison of the seeplex reverse transcription pcr assay with the rmix viral culture and immunofluorescence techniques for detection of eight respiratory viruses high-throughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses the tennessee children's respiratory initiative: objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases osterhaus ad: a previously undescribed coronavirus associated with respiratory disease in humans the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction clinical characteristics and -day outcomes for influenza a (h n ) evaluation of a multiplexed pcr assay for detection of respiratory viral pathogens in a public health laboratory setting multicode-plx system for multiplexed detection of seventeen respiratory viruses comparison of reverse transcription-pcr with tissue culture and other rapid diagnostic assays for detection of type a influenza virus nucleic acid amplification tests for detection of respiratory viruses real-time pcr in the microbiology laboratory comparison of the eragen multi-code respiratory virus panel with conventional viral testing and real-time multiplex pcr assays for detection of respiratory viruses the classification of viruses infecting the respiratory tract simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections linkage between the journal and quality control molecular diagnostics (qcmd) real-world comparison of two molecular methods for detection of respiratory viruses james gern is on the scientific advisory board for and has stock options for v biosciences, has consulted for and has stock options for eragen biosciences, has consulted for synairgen and centocor, and has received research support from astrazeneca and merck. kathryn m. edwards receives research funding from sanofi-pasteur, wyeth, novartis, and csl. marie r griffin has received grant support from medimmune and wyeth. john v. williams has served as a consultant for medimmune, novartis and serves on the scientific advisory board of quidel. submit your next manuscript to biomed central and take full advantage of: key: cord- -dzin qb authors: zhang, wei; chen, keren; guo, yang; chen, yaosheng; liu, xiaohong title: involvement of prrsv nsp and nsp in the autophagy process date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: dzin qb background: autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. methods: lc turnover and the proteins in the endoplasmic reticulum (er) stress pathway were investigated using western blot analysis. the formation and degradation of autophagosomes were detected using immunofluorescence staining. results: autophagy was activated by porcine reproductive and respiratory syndrome virus (prrsv) nsp , nsp and nsp , which are two transmembrane proteins and an rna-dependent rna polymerase, respectively. the formation of autophagosomes was induced by nsp and nsp and developed from the er; the fusion of these autophagosomes with lysosomes was limited. although nsp and nsp are er transmembrane proteins, these proteins did not activate the er stress signaling pathways. in addition, the cytoplasmic domain of nsp plays a pivotal role in activating autophagy. conclusions: the data presented in this study reveal an important relationship between prrsv nsps and autophagy and provide new insights that improve our understanding of the involvement of prrsv nsps in the autophagy process. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome virus (prrsv- ) is a member of the genus arterivirus, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv) [ ] . prrsv infection leads to severe morbidity and mortality worldwide; the main clinical manifestations are reproductive disorders, such as miscarriage, stillbirth, and the delivery of mummified fetuses in pregnant sows, as well as respiratory symptoms in pigs of various ages (particularly piglets) [ , ] . prrsv is an enveloped, single-stranded positive-strand rna virus with a genome of approximately . kb that includes at least open reading frames (orfs); orf a and orf b encode nonstructural proteins (nsps), and orf -orf encode structural proteins [ , ] . these nsps are involved in viral replication and play key roles in regulating genomic transcription [ ] . among these nsps, nsp and nsp are predicted to be transmembrane proteins and nsp possesses viral rna-dependent rna polymerase activity [ ] . prrsv is usually grown in marc- cells, which originated from monkey kidney epithelial cells. autophagy occurs in eukaryotic cells and is a process by which cell homeostasis is maintained through the degradation of proteins and organelles [ , ] . generally, autophagy is triggered in cells stimulated with external factors, such as starvation or viral infection; the marker of autophagy is the formation of autophagosomes [ ] . the integrated autophagy course primarily comprises three processes: first, the formation of autophagosomes; then, the fusion of autophagosomes with lysosomes; and last, degradation of the cargo in lysosomes. the first step of the autophagy process is that a membrane possibly derived from the endoplasmic reticulum (er) elongates, becomes curled, and then surrounds part of the cytoplasm, followed by the degradation of organelles, proteins and other cellular components. in this process, two proteins, namely, atg and atg , form complexes via ubiquitin-like conjugation [ , ] . furthermore, atg interacts with the atg -atg complex to form the atg -atg -atg protein complex, which plays a significant role in forming autophagosomes [ ] . finally, lysosomes fuse with mature autophagosomes to form autophagolysosomes; the contents of the autophagosome are degraded, and the substances inside the autophagolysosomes are delivered to the cytoplasm for transformation into amino acids and energy for cytoplasmic metabolism [ ] . generally, autophagy is accompanied by er stress, which is induced by the accumulation of unfolded or misfolded proteins in the er [ ] . the er is the main site of protein synthesis, lipid production and calcium ion storage in eukaryotic cells. a variety of proteins in the er must be folded, assembled, processed, packaged and transported to the golgi apparatus [ ] . once the cells are stimulated with internal and external factors, the balance between er morphology and function is affected by the changes in molecular biochemistry. protein processing and transport are blocked, and a large number of unfolded or misfolded proteins accumulate in the er, leading to the unfolded protein response (upr) [ ] [ ] [ ] , which operates through three signaling pathways that play significant roles in regulating autophagy during viral infection [ , ] . the er stress response was recently shown to be induced by prrsv infection, leading to the activation of jnk signaling pathways [ ] . in the past decade, a number of studies have reported a connection between autophagy and viral proteins [ , ] . the hcv polymerase ns b was recently reported to colocalize with ns b and interact with atg to initiate autophagosome formation [ ] . in addition, hbx promotes autophagy and prevents the fusion of autophagosomes with lysosomes to maintain the accumulation of solutions in the autophagosomes [ , ] . recent studies examining the relation between autophagy and prrsv revealed that prrsv induced incomplete autophagy and that autophagy enhances the replication of prrsv [ ] [ ] [ ] . additionally, prrsv infection promotes mitochondrial fission and activates apoptosis [ ] [ ] [ ] ; however, few studies have examined the relations between prrsv nsps and autophagy to date. in this study, we detected the involvement of prrsv nsps in autophagy in transfected cells for the first time. prrsv nsp , nsp and nsp induce the formation of autophagosomes. additionally, nsp and nsp are er transmembrane proteins, but these two nsps trigger limited er stress, and the autophagosomes induced by these two nsps are derived from the er and accumulate autophagic cargo to prevent fusion with lysosomes. furthermore, an nsp truncation mutant consisting of only the hydrophobic domains did not induce the formation of autophagosomes. our findings revealed the important functions of prrsv nsps in autophagy. cells of the african green monkey kidney epithelial cell line marc- purchased from atcc were cultivated in dulbecco's modified eagle's medium (dmem, invitrogen) supplemented with % fetal bovine serum (fcs) at °c in a % co atmosphere. hbss (hank's balanced salt solution) was purchased from sigma and can induce autophagy. the prrsv strain ch- a (the first prrsv strain isolated from china) was provided by dr. guihong zhang at the south china agricultural university, china. cellular dna was stained with ′, ′-diamidino- -phenylindole dihydrochloride (dapi), and mitochondria were stained with mitotracker green. rabbit anti-lc , anti-calnexin, and anti-gapdh; mouse anti-atg ; alexa fluor -conjugated anti-rabbit igg; alexa fluor -conjugated anti-mouse igg; and alexa fluor -conjugated anti-mouse igg secondary antibodies were purchased from cell signaling technology (beverly, ma). a mouse anti-mcherry antibody was obtained from abcam (ab ). a prrsv nucleocapsid (n) antibody was purchased from jeno biotech inc. (chuncheon). the mouse monoclonal anti-dsrna antibody was purchased from scicons (hungary). viral rna was isolated with trizol (invitrogen), and cdnas were obtained using a reverse transcription kit (promega). pcr primers were designed to amplify the prrsv nsp transcripts (additional file : table s ) using the prrsv ch- a cdna. the pcr fragments were cloned into a pmcherryn (clontech, , ) expression vector. the truncated prrsv nsp mutant was constructed using a pcr primer pair (additional file : table s ). all multiple cloning digestion sites were selected for digestion with xhoi and bamhi. marc- cells were transfected with μg of the constructed plasmids using lipofectamine (invitrogen). marc- cells were cultured on glass coverslips, followed by transfection with the individual plasmids for h. after three washes with phosphate-buffered saline (pbs), cells were fixed with % paraformaldehyde (pfa) for min at room temperature. after three washes with pbs, the cells were permeabilized with . % triton x- for min and blocked with % bovine serum albumin (bsa) in pbs for one hour at room temperature. next, the coverslips were incubated with primary antibodies (diluted : ) in pbs containing % bsa for h, followed by an incubation with an alexa fluor-conjugated secondary antibody (diluted : ) for h at room temperature in the dark. after three washes with pbs, cellular nuclei were stained with dapi in the dark for min at room temperature. next, the coverslips were washed with pbs, mounted on the microscope slides with antifade mounting medium, and observed under a leica tcs sp confocal microscope. cells were washed with pbs, and total protein was obtained using sds cell lysis buffer ( % sds and mm tris, ph . ) containing mm phenylmethylsulfonyl fluoride (pmsf) and heating for min. a volume of lysate containing μg of protein was separated on % acrylamide gels in a bio-rad system. then, proteins were transferred to a polyvinyl difluoride (pvdf) membrane and blocked with % bsa in tris-buffered saline including tween (tbst) for min. after blocking, the pvdf membrane was incubated with the primary antibody for h, followed by incubation with the hrp-conjugated secondary antibodies. images were captured using an image station mm pro system and image station mm pro software. cells were collected, and total rna was extracted using trizol (invitrogen). two micrograms of rna was obtained with the reverse transcription kit as described above. the synthesized cdnas were amplified with specific primers (additional file : table s ). the final products were visualized on a . % agarose gel that was electrophoresed at v for min. the pearson correlation coefficient per cell was quantified using image-pro plus software. data are presented as means ± standard errors. statistical significance was determined by performing student's t-tests. p values < . were considered statistically significant. the gfp-lc plasmid, which expressed the lc protein tagged at its n terminus with the fluorescent protein gfp, was used to monitor the formation of autophagosomes by indirect immunofluorescence [ ] . gfp-lc was transfected into cells for h, and transfection efficiency was - %. cells were then infected with prrsv ch- a. at h.p.i., the infected cells were fixed, and gfp-lc puncta were observed to assess the formation of autophagosomes. as shown in fig. a and b, compared to the accumulation of gfp-lc puncta in the cytoplasm of mock-infected cells, the accumulation of these puncta in the cytoplasm of hbss-treated and prrsv-infected cells suggested that prrsv induced the formation of autophagosomes. lc conversion is a hallmark of autophagy; therefore, the conversion of lc was assessed by immunoblotting and the levels of lc ii/ lc i were examined to assess the induction of autophagy. marc- cells were infected with prrsv ch- a at h.p.i. or were cultured with hbss for h as a positive control. as shown in fig. c , compared to the lc ii/ lc i ratio in the mock-infected cells, the ratio was increased in the infected marc- cells. we explored whether prrsv dsrna and n proteins were associated with autophagosomes using confocal microscopy to identify whether the autophagosomes induced by prrsv were related to viral replication or assembly. as depicted in fig. d , the majority of the lc protein was colocalized with dsrna and n proteins, indicating that these autophagosomes provide the site for prrsv replication and assembly. prrsv non-structural proteins play an important role in virus replication and assembly and use the substances in the cells to influence cell life activities. because prrsv induced the formation of autophagosomes, we further explored which prrsv nsps played important roles in this process. eukaryotic expression vectors carrying the nsp cdnas with an n-terminal mcherry tag were constructed and transfected into marc- cells (additional file : figure s ). as shown in fig. a and b, the gfp-lc puncta accumulated in nsp -mcherry-, nsp -mcherry-and nsp -mcherry-transfected cells. nsp is an rna-dependent rna polymerase (rdrp) that plays important roles in viral transcription and replication, and nsp and nsp are predicted to be transmembrane proteins; these proteins are anchored on the cytoplasmic membrane and are part of the membrane-bound replication and transcription complex. furthermore, lc levels were detected using immunoblotting to determine the effects of the two transmembrane proteins on autophagy. p /sequestosome- is a protein that can bind to lc as a scaffold protein or a signaling adapter and may be increased at the beginning of autophagy process and degraded gradually. based on the data presented in fig. c , immunoblotting and immunofluorescence assay showed that the expression of the p protein was increased, indicating that nsp and nsp of prrsv induced the formation of autophagosomes. as shown in fig. d , a higher lc ii/ lc i ratio was observed in nsp -mcherry-and nsp -mcherry-transfected cells than in mock-infected cells. we identified some of the effects of these two transmembrane proteins on cell autophagy to determine whether the nsps affected cytoplasmic membrane fusion. the autophagosomes induced by nsp and nsp are derived from the er generally, in the early stage of autophagy, atg complexes with atg and atg l to form the atg -atg -atg l complex [ ] , and this complex attaches to nascent phagophores, which are the precursors of mature autophagosomes that are involved in regulating membrane morphology changes [ ] . therefore, endogenous atg was confirmed at the site of immature autophagosomes. autophagosome membranes have been shown to originate from the mitochondria [ ] or er [ ] . cells transfected with nsp -mcherry and nsp -mcherry were fixed and stained for atg and calnexin, an er marker. the value of overlap of the two different channels was calculated using pearson's correlation coefficient, and a value exceeding . was determined to indicate colocalization. as shown in fig. a , atg puncta were arranged in reticular structures and colocalized with calnexin in the fig. the distribution of autophagy proteins in prrsv-infected marc- cells. a marc- cells were transfected with gfp-lc plasmids and cultured with either dmem or hbss media for h or were infected with prrsv ch- a for h. fixed cells were observed under a fluorescence microscope. nuclei were stained with dapi (blue), and virions were stained with an antibody against the prrsv-n protein (red). scale bars: μm. b statistical analysis of the number of gfp-lc puncta in mock, hbss-treated or prrsv-infected cells; the number represents gfp-lc puncta per cell; data are presented as means ± sd, n = . c lc conversion in marc- cells. marc- cells were mock infected, infected with prrsv for h or cultured in hbss media. cells lysates were subjected to immunoblotting. the ratio of lc ii/lc i reflects the level of autophagy. d marc- cells were infected with prrsv for h, and fixed cells were observed under a fluorescence microscope. nuclei were stained with dapi (blue); dsrna and prrsv-n are labeled in red, and endogenous lc is labeled in green. scale bars: μm cells expressing nsp and nsp . combined with the subsequent findings that prrsv nsp and nsp were localized on the er, these results suggested that the atg puncta were derived from the er. little colocalization of the fluorescently labeled atg proteins with mitotracker green, a mitochondrial marker, was observed in cells transfected with nsp -mcherry and nsp -mcherry (fig. b) . therefore, the autophagosomes induced by prrsv nsp and nsp originate from the er but not from the mitochondria. after mature autophagosomes are formed, they fuse with lysosomes and form autolysosomes to degrade the insoluble substances inside the autophagosomes. fig. the formation of autophagosomes induced by prrsv nsp and nsp . a marc- cells were transfected with gfp-lc and plasmids encoding the individual prrsv nsps; each nsp was cloned into an mcherry-n plasmid, and cells transfected with mcherry-n were used as a negative control. twenty-four hours after transfection, cells were harvested and visualized under a fluorescence microscope. nuclei were stained with dapi, and the number of gfp-lc puncta, which is related to autophagy, was analyzed using graphpad software. scale bars: μm. b statistical analysis of the number of gfp-lc puncta in cells expressing mcherry-n and each nsp; the numbers represent gfp-lc puncta per cell; data are presented as means ± sd, n = . c cells were transfected with mcherry nsp -mcherry and nsp -mcherry for h; the endogenous p protein was stained with a green fluorophore. cell lysates were subjected to immunoblotting. scale bars: μm. d lc protein conversion. marc- cells were mock infected; treated with hbss for h or transfected with mcherry, nsp -mcherry, or nsp -mcherry; and then harvested and lysed. extracts were analyzed by western blotting using an anti-lc antibody. the ratio of lc ii/lc i reflects the level of autophagy we labeled nsp -and nsp -transfected cells with lysosome-associated membrane protein (lamp ), a lysosomal marker, and the mature autophagosome marker lc to investigate the fate of autophagosomes and to obtain a better understanding of the role of these autophagosomes induced by the two nsps. as shown in fig. a , an overlap of lc and lamp was observed in serum-starved marc- cells. as shown in fig. b , in nsp or nsp transfected cells, lamp failed to colocalize with lc , and the pearson correlation coefficient was much lower than that in serum-starved cells, suggesting that lysosomes did not fuse with the autophagosomes induced by the two nsps, as with prrsv-infected cells. these results suggest that the autophagosomes induced by nsp and nsp are derived from the er, but autophagosome-lysosome fusion was limited. nsp and nsp do not induce er stress prrsv nsp and nsp are predicted to be transmembrane proteins, and we studied which organelle membrane is the attachment site of these two integral transmembrane proteins. as shown in fig. a and b, prrsv nsp -mcherry and nsp -mcherry only colocalized with calnexin but not with mitotracker green. thus, prrsv nsp and nsp are integral transmembrane proteins of the er but not the mitochondria. the activation of autophagy is always affected by er stress, which is due to protein processing dysfunctions; the c/ebp homologous protein (chop) is overexpressed [ ] , and the xbp mrna is spliced by phosphorylated ire . prrsv triggers the activation of the ire α and perk branches of the upr, suggesting that chop overexpression is activated by the perk pathway and that the autophagosomes induced by prrsv nsp and nsp are derived from the er; therefore, we examined chop expression using immunoblotting, and we determined the splicing of the xbp mrna using pcr. as shown in fig. c , compared to the expression of chop in mock-infected cells, this protein was noticeably upregulated in prrsv-infected cells; however, the expression of chop in nsp -mcherry-and nsp -mcherry-transfected cells was not significantly fig. autophagosomes originated from the er but not mitochondria. a marc- cells were transfected with nsp -mcherry and nsp -mcherry for h; atg is labeled in green, and nuclei were stained with dapi (blue). scale bars: μm. statistical analysis of the pearson correlation coefficient for calnexin and atg . the pearson colocalization coefficient is presented as the ratio of punctate signals of calnexin that were positive for atg . b nsp -mcherry and nsp -mcherry were expressed in marc- cells. twenty-four hours after transfection, cells were cultured with mitotracker green, a marker of mitochondria, and were then fixed and immunostained with an anti-atg antibody (pink). nuclei were stained with dapi (blue). scale bars: μm. statistical analysis of the pearson correlation coefficient for mitotracker green and atg . the colocalization coefficient is presented as the ratio of fluorescent signals of mitotracker green that were negative for atg changed. thus, the involvement of prrsv nsp and nsp in autophagy was not regulated by an er stress-dependent pathway. the cytoplasmic domain is required for prrsv nsp induced autophagy prrsv nsp and nsp are predicted transmembrane proteins. as shown in fig. a, nsp consists of hydrophobic transmembrane domains and a hydrophilic cytoplasmic domain and nsp consists of hydrophobic transmembrane domains. a deletion mutant in which nsp consisted of only the hydrophobic domains was constructed to detect whether the hydrophilic cytoplasmic domain of nsp , which is out of range of the membrane, affects the formation of autophagosomes. as illustrated in fig. b , the nsp mutant was successfully constructed, and the truncated protein exhibited a lower molecular weight than the normal nsp protein. furthermore, as shown in fig. c , along with the normal nsp protein, the mutant nsp protein was localized on the er but did not induce the formation of autophagosomes. these results reveal that the hydrophilic cytoplasmic domain of nsp plays an important role in inducing autophagy. knowledge about virus-induced autophagy has gradually increased [ ] . however, the relationship between each prrsv protein and autophagy has not been elucidated. as shown in the present study, the induction of autophagy by prrsv nsp and nsp contributed to the formation of autophagosomes derived from the er, and the mature autophagosomes were not degraded by fusion with lysosomes. prrsv nsp and nsp are er transmembrane proteins, but these multiple processes of autophagy were not induced by er stress. moreover, a deletion mutant of nsp revealed that the transmembrane domains are crucial for inducing the formation of autophagosomes. our findings offer new explanations for the activation of autophagy by prrsv nsps. autophagy plays an important role in both the innate and adaptive immune responses, both of which eliminate intracellular microbes [ ] . however, many pathogens that invade cells induce the formation of double-membrane autophagosomes to facilitate their own replication [ ] . almost all viruses induce the formation of autophagosomes, and autophagosomes provide the site for viral replication and assembly [ ] . additionally, individual nonstructural proteins or structural proteins may perform viral functions, such as inducing apoptosis or necrosis. in the present study, prrsv ch- a induced the formation of autophagosomes in the infected cells; these autophagosomes are purported to be the site of viral replication. viral nonstructural proteins play important roles in the replication of the viral rna and in the synthesis of viral particles. in virus-infected cells, the virus interferes with and destroys the normal metabolism of the host cell, causing cell death or apoptosis [ ] . in many studies, viral nonstructural proteins have been shown to play important roles in virus-induced autophagy. in hcv-infected cells, ns / a blocks the rig-i signaling pathway in the early stages of autophagy and modulates the binding of mitochondria-associated immunity-associated gtpase family m (irgm) to promote autophagy [ ] . similarly, jev ns targets irgm to modulate autophagy [ ] , and cbv protein b, whose c-terminal sequence plays a decisive role in autophagy, rearranges the cell membrane [ ] . in our study, gfp-lc puncta accumulated in nsp -mcherry-, nsp -mcherry-and nsp -mcherry-transfected cells and p expression was increased, indicating that nsp , nsp and nsp induced the formation of autophagosomes. furthermore, we detected an increase in the ratio of lc ii to lc i in cells transfected with nsp and nsp , along with an increased expression of p , confirming that nsp and nsp induce autophagy. generally, the process of autophagy includes two steps: the formation of autophagosomes and the degradation of autophagosomes. in the early stage of autophagy, atg interacts with atg and atg to form the atg -atg -atg l complex; this complex is localized in phagophores, which are the precursors of autophagosomes. in addition, the majority of autophagic degradation occurs when autophagosomes envelope cellular substances, which are delivered to the lysosomes for degradation of the contents. in the present study, a marker of phagosomes, which are the precursor of autophagosomes, namely, atg , was colocalized with calnexin, an er marker, but not with mitotracker, suggesting that the autophagosomes induced by nsp and nsp were derived from the er but not from the mitochondria. moreover, autophagy is a complex process. after prrsv infects cells, it incompletely induces autophagy and inhibits the fusion of autophagosomes with lysosomes. this inhibition leads to the accumulation of autophagosomes and enhances prrsv replication. in our study, lc was not colocalized with lamp in nsp -and nsp -transfected cells, suggesting that the fig. a nsp and nsp were located in the er but not the mitochondria. nsp -mcherry and nsp -mcherry were expressed in marc- cells; the er was stained with an antibody against endogenous calnexin (green), and mitochondria were stained with mitotracker green. scale bars: μm. b statistical analysis of the pearson correlation coefficients for calnexin/mitotracker and nsp /nsp . the colocalization coefficient is presented as the ratio of fluorescent signals of calnexin that were positive for nsp /nsp . the colocalization coefficient is the ratio of fluorescent signals of calnexin that were negative for nsp /nsp . c chop expression was examined in mock-infected; prrsv-infected; and mcherry-, nsp -mcherry-, or nsp -mcherry-expressing marc- cells using immunoblotting. the intensities of chop bands were normalized to gapdh. the splicing of the xbp mrna was examined using pcr; xbp u is the unspliced mrna, and xbp s is the spliced mrna mature autophagosomes induced by nsp and nsp were not fused with lysosomes. our findings provide evidence that the membranes of autophagosomes induced by nsp and nsp were derived from the er and inhibited autophagosome fusion with lysosomes. nsp and nsp are two transmembrane proteins that appear to play a role in membrane rearrangement. prrsv nsp and nsp were localized to the er, but not the mitochondria, in the present study, suggesting that the two nsps are transmembrane er proteins but not transmembrane mitochondrial proteins. in a recent study, er stress was activated in prrsv-infected cells. additionally, researchers previously believed that autophagy activation is accompanied by the activation of the er stress pathway. the early er stress response involves the splicing of the xbp mrna and the expression of chop. in the present study, we confirmed that the splicing of the xbp mrna was not noticeably altered and chop expression exhibited a slight increase, indicating that autophagy was not activated by er stress in prrsv nsp -and nsp -transfected cells. because prrsv nsp contains transmembrane domains and a hydrophilic cytoplasmic domain, we deleted the cytoplasmic domain and revealed that the activation of autophagy requires complete nsp protein. in conclusion, prrsv nsp and nsp , which are er transmembrane proteins, induce the formation of autophagosomes. although these autophagosomes are derived from the er and are not degraded by lysosomes, the activation of autophagy by the two nsps does not involve er stress. the hydrophilic cytoplasmic domain of prrsv nsp plays a key role in the activation of autophagy. overall, our findings provide new insights into the connection between autophagy processes and prrsv nsps. the nsp hydrophilic domain is required to activate autophagy. a schematic of the prrsv nsp , nsp and mutant nsp protein structures. b mcherry was detected by immunoblotting in nsp -and mutant nsp -expressing cells c marc- cells were co-transfected with plasmids expressing wildtype/mutant prrsv nsp and gfp-lc for h. fixed cells were detected using fluorescence microscopy. gfp-lc puncta represent mature autophagosomes; nuclei were stained with dapi (blue). scale bars: μm prrsv, the virus porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states prrsv structure, replication and recombination: origin of phenotype and genotype diversity nidovirales: a new order comprising coronaviridae and arteriviridae the structural biology of prrsv overview: replication of porcine reproductive and respiratory syndrome virus cell biology -autophagy as a regulated pathway of cellular degradation the role of autophagy during the early neonatal starvation period autophagosome formation: core machinery and adaptations molecules and their functions in autophagy the atg -atg conjugate has a novel e -like activity for protein lipidation in autophagy formation of the similar to -kda apg -apg center dot apg multimeric complex, mediated by apg oligomerization, is essential for autophagy in yeast guidelines for the use and interpretation of assays for monitoring autophagy regulation mechanisms and signaling pathways of autophagy endoplasmic reticulum is the sorting core facility in the golgi-lacking protozoan giardia lamblia the unfolded protein response: from stress pathway to homeostatic regulation signal integration in the endoplasmic reticulum unfolded protein response endoplasmic reticulum stress: cell life and death decisions endoplasmic reticulum stress in disease pathogenesis er stress and diseases involvement of unfolded protein response, p and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate coronavirus nsp restricts autophagosome expansion autophagy protein atg interacts transiently with the hepatitis polymerase (ns b) early during infection hepatitis b virus x protein induces autophagy via activating death-associated protein kinase hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication hu hb: autophagy sustains the replication of porcine reproductive and respiratory virus in host cells porcine reproductive and respiratory syndrome virus triggers mitochondrial fission and mitophagy to attenuate apoptosis interplay of autophagy and apoptosis during prrsv infection of marc cell autophagy postpones apoptotic cell death in prrsv infection through bad-beclin interaction apg p is required for the function of the apg p-apg p conjugate in the yeast autophagy pathway the role of atg proteins in autophagosome formation mitochondria supply membranes for autophagosome biogenesis during starvation autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum chop is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum the role of autophagy in host defence against mycobacterium tuberculosis infection an integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly how positive-strand rna viruses benefit from autophagosome maturation the crosstalk between autophagy and apoptosis: where does this lead? hepatitis c virus and autophagy capsid, membrane and ns are the major viral proteins involved in autophagy induced by japanese encephalitis virus protein b of coxsackievirus b induces autophagy relying on its transmembrane hydrophobic sequences the authors wish to acknowledge professor guihong zhang from the south china agricultural university for her donation of experimental materials and her influential guidance to the authors. this study was funded by grants from the natural science foundation of guangdong province ( a ), the national natural science foundation of china ( ) and the guangdong sailing program ( yt h ). additional file : table s . table primers used availability of data and materials not applicable. the major contributions of w.z. were in the writing of the manuscript, designing the experiments and performing the experiments. y.g, k.c, y.c. and x.l. composed the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.received: july accepted: january key: cord- -ba wq t authors: sias, catia; salichos, leonidas; lapa, daniele; del nonno, franca; baiocchini, andrea; capobianchi, maria rosaria; garbuglia, anna rosa title: alpha, beta, gamma human papillomaviruses (hpv) detection with a different sets of primers in oropharyngeal swabs, anal and cervical samples date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: ba wq t background: recent studies have shown a -fold increase of oropharyngeal cancer in the presence of hpv, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical district, many novel β and γ types have been isolated in this anatomical site suggesting a wide tropism range. currently, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and it remains unknown which hpv types should be included in hpv screening programs. our goal was to assess hpv prevalence in oropharyngeal, anal, and cervical swabs using different sets of primers,which are able to amplify α, β, γ hpv types. methods: we analysed the presence of hpv dna in oropharyngeal (n = ), anal (n = ), cervical specimens (n = ) from hiv positive and negative patients using fap / and my / primers. all untyped strains were genetically characterized through pcr amplification and direct sequencing of partial l region, and the resulting sequences were classified through phylogenetic analysis. results: hpv prevalence was . % in oropharyngeal swab samples, including infections with multiple hpv types ( . %). hpv prevalence in this anatomical site was significantly associated with serostatus: . %in hiv positive and . % in hiv negative patients (p < . ). unclassified types were detected in specimens. in our analysis, we did not observe any difference in hpv (α, β, γ) prevalence between men and women. overall, β species were the most frequently detected . %. when using anal swabs, for hiv positive patients, β genus prevalence was % and γ genus was . % including unclassified types. in cervical samples from hiv positive women ( hpv negative and positive by my / pcr), only one sample was positivite for β( ) species ( . %) using fap primers. six of the untyped strains clustered with sequences from species , , , , of γ genus. four sequences remained unclassified. finally, β and γ hpv prevalence was significantly lower than their respective hpv prevalence as identified by the luminex system in all anatomical sites that were analyzed in previous studies. conclusion: this study provides new information about viral isolates present in oropharyngeal site and it will contribute to improve the monitoring of hpv infection. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. human papillomavirus (hpv) persistent infections are considered the primary cause of ano-genital cancer, where greater than % of cervical cancer and more than % of anal cancer contain hpv dna [ , ] . throughout the past years, several studies have suggested the link between hpv infection and other epithelial cancers, including cutaneous and oropharyngeal cancers. oropharyngeal cancers are often referred to as "head and neck cancer" (hnc), and mainly include squamous-cell carcinoma which occurs in the oral cavity, base of tongue, tonsils, adenoids pharynx and larynx. recent studies on hpv infection in oral exfoliated cells have shown that there is a -fold higher risk for oropharyngeal cancer in the presence of virus [ ] [ ] [ ] . the highest prevalence (up to %) for these hpv associated cancers has been found in the tonsillar cancers sub-set [ ] [ ] [ ] . since hpv presented a global prevalence in oropharyngeal squamous cell carcinoma (opscc) and oral squamous cell carcinoma (oscc) of . and . % respectively [ ] , iarc categorized this genotype as a risk factor with for the pathogenesis of both cancers [ ] . additionally, other hpv types have also been linked with these cancers, including hpv , , and [ ] including β and ɣ types [ ] . furthermore, while α-hpv detection seems to be rare in oral cavity in comparison to anal or cervical districts [ ] , many novel β and γ types have been isolated in oral cavity [ ] suggesting a wide tropism range of these genera [ ] . at the same time, different studies on hpv prevalence provided contradicting result for β and ɣ types not only in oropharyngeal site, but also in cervical and anal anatomical sites [ ] [ ] [ ] [ ] [ ] [ ] . to date, there are no guidelines recommending hpv oral cavity screening as a mandatory test, and no hpv dna test has been approved for hpv detection in oral cavity. in addition, it remains unknown which hpv types should be included in oral cavity screening. agalliou [ ] highlighted the link between β, γ hpv types and oral cancers, assessing the presence of hpv types which are not generally detected with commercial assays in cervical cancer screening. even though different methods have been used to detect novel hpv types, the association between new β and γ hpv types and oral cavity cancer has not yet been established. in this study, we analyzed the presence of hpv dna in oral, anal, and cervical specimens collected from hiv positive and hiv negative individuals, living in the same geographic area (regione lazio) by using my / [ , ] fap / primers [ ] . these primers, both targeting highly conserved region within l orf, are considered broad range pv primers and allow the detection of the great majority of already known and officially recognized hpv types. my / had been used in α hpv detection in cervical sites, where they showed good sensitivity in alpha hpv types amplification [ ] , while fap primers officially recognized hpv from α-pv, β-pv, and ɣ-pv genera, and might detect potentially new types [ ] [ ] [ ] [ ] . this property is particularly relevant, since ɣ and β papillomaviruses have been already identified in several anatomic sites [ , ] . granted that that hpv prevalence is significantly higher among hiv infected people and at multiple anatomic sites [ ] [ ] [ ] [ ] we included both hiv positive and hiv negative people in our study in order to also verify whether β and ɣ hpv positivity is influenced by host immune status. this is a retrospective study carried out at laboratory of virology inmi l spallanzani on residual oropharyngeal samples collected for respiratory virus detection, anal and cervical swabs collected for hpv testing in diagnostic routine. the hospital ethics committee approved the protocol. date of birth, date of swabs sampling, hiv serostatus were recorded from institutional database. in hiv positive patients, the most recent cd t cell count (± month from the date of sample collection) and hiv rna viral load (± month from the date of sample) with a detection limit of cp/ml (abbott molecular inc., des plaines, il, usa) were used to correlate clinical features and hpv positivity. oropharyngeal swabs -oropharyngeal samples were collected as following: the nylon-flocked tip was rotated - -times against right and left buccal mucosa, palatine, tonsils, upper and lower pharynx area. the swab was then plunged and stirred in . ml dmem medium with streptomycin and ampicillin. specimens were refrigerated within - h after collection until processing. anal swabs -we retrospectively analysed anal swabs previously tested by my / primers and typed by clart hpv clinical array or sanger sequencing (see hpv detection and typing section, below). one hundred samples belonged to hiv positive women ( hpv positive by my / pcr and negative by my / pcr), anal swabs were collected from men who have sex with men (msm). all msm specimen resulted positive in my / pcr. cervical swabs -a total of cervical swabs ( hpv negative and hpv positive, assigned by my / pcr) were considered in this study. all samples belonged to hiv positive women. general characteristics of hiv infected patients are presented in additional file : table s . oropharyngeal swabs were removed from the medium which we divided in two parts: half part was used for detecting respiratory viruses panel (influenza-a, b, rsv, rhinovirus, coronaviruses, metapneumovirus, adenovirus) and the other half was employed in testing hpv dna, having been stored at − °c until use. before nucleic acid extraction, all specimens were pre-treated. briefly, μl of clinical material was digested with μl proteinase k solution (qiagen, hilden germany) and lysed with al lysis buffer (qiagen, hilden, germany) at °c for min. nucleic acid extraction was done with a magnetic bead-based automated platform (qiasymphony, hilden, germany) in accordance with the manufacturer's instructions. nucleic acids were eluted in μl of ave elution buffer (qiagen, hilden, germany). nucleic acid from anal and cervical swabs were extracted as described elsewhere [ , ] . samples that were β-globin negative were excluded from the study [ , ] . ten μl of eluted nucleic acids were employed for evaluating the presence of hpv types by using my ( 'cgtccmarrggawactgatc ') and my ( 'gcmcagggwcataayaatgg ') [ , ] pcr and faststart dna polymerase (roche diagnostics gmbh, mannheim, germany). the pcr assay conditions were: °c for min, then cycles (denaturation °c/ s, annealing °c/ s, and extension °c/ min). one last step for extension was employed at °c for min. fifteen μl of the pcr products were mixed with loading solution in . % agarose gel electrophoresis stained by ethidium bromide and run for min at v. hpv genotyping of positive samples was conducted using genomica clinical hpv array (genomica, madrid, spain). clart hpv clinical array hpv is able to detect: , , , , , , , , , , , , , , , , , , , all adequate samples were retested using fap ( 'taacwgtiggicayccwtatt ') and fap ( 'ccwatatcwvhcatitciccatc ') primers [ ] able to detect α, β and γ hpv types. faststart dna polymerase were used in this assay condition: min at °c, cycles (denaturation °c/ s, annealing °c / s, and extension °c min) followed by a final extension step at °c for min. pcr products were also run on agarose gel. positive samples with fap set of primers and those positive with my / pcr protocol, but negative in genomica typing assay were purified and sequenced on the automated abi prism instrument, by using a bigdye terminator cycle sequencing kit (applied biosystems, warrington, uk). hpv sequences were aligned using mafft [ ] using the l-ins-i method. after visual inspection using seaview [ ] we removed sample sequence q and q were excluded from the alignment because they represented mixed infection and the sanger sequence interpretation was not optimal. reference sequences hm _hpv and gu _hpv were also excluded from the alignment. then, we further implemented gblocks [ ] to remove ambiguous positions using the least stringent options. to infer a maximum likelihood tree, we implemented raxml [ ] using a gtrcat model, with bootstrap replicates. tree visualization was achieved using figtree [ ] . finally, pairwise similarity matrices were constructed using blast. oropharyngeal swabs-a total of oropharyngeal swabs with β-globin positive signal were considered in this study. hpv prevalence was . % ( / ), including infections with multiple hpv types ( / , . %) ( table ). my / pcr gave positive results in samples. however, a blast search against genbank indicated that amplified fragments were identical to human sequences ( / , %), thus true hpv positive rate was estimated at . %. out of , three samples were identified as α and ten as β types. alpha-types were detected in two hiv positive patients ( / , . %), and in hiv negative patient. hpv α-types, and , were detected in hiv positive subjects ( / , . %) after being typed by clart hpv clinical array, while hpv was amplified in hiv negative subject ( / , . %); it was typed by sanger sequencing. all β hpv types belonged to β species (table ) , and hpv was the most frequent type ( / ), both in hiv positive and hiv negative patients; samples were identified as positive by fap pcr. no specific amplicons were observed. seven fap-detected hpv types were found in samples that also gave positive results with my /my pcr (table ) . types β were the most represented (n = ), while one sample was identified as γ type (hpv , γ ). additionally, one mixed infection (q ) and untyped hpv strains were also detected. among β species, the most frequent types were hpv (n = , . %) and hpv (n = , . %). three multiple infections harboured β and β types, whereas β types and untyped strains were observed in other multiple infections. no multiple infection harboured α with β or γ species (table ) . overall, β species were most prevalent (n = / , . %). hpv distribution differed significantly when the number of hpv β, γ-types were considered in hiv positive and hiv negative subjects: ( / , . %) vs ( / , . %) respectively (chi square test, p < . ). hiv positive people with hpv in oropharyngeal swabs showed a mean cd t cell count of . ± . . among hiv positive patients with detectable hpv, / had no detectable hiv rna in plasma and the other patients (n = ) showed a mean rna copies/ml . ± . . hpv prevalence rates were compared for statistical significance, using a chi squared test, in men and women both hiv positive and hiv negative. however, no difference was observed (chi square test, p > . ). anal swabs-eighty-six anal swabs from msm resulted positive by my / pcr, and anal swabs from women ( hpv positive by my / pcr and negative by my / pcr) were retested with fap primers. among msm anal swabs, samples showed single hpv infection by clart array testing, and patients harboured multiple hpv infection with at least one high risk (hr) type. in . % of samples we observed a clinically evident anal pathology, including subjects with anal intraepithelial neoplasia (ain) ii, patients with ain i. among hpv positive women, harboured a single infection (ain ii, n = ; ain i, n = ); atypical squamous cells of undetermined significance (asc-us), n = ; normal, n = ) and samples showed hpv multiple infection, all with at least hr type ( %) (ain ,n = ; ain i, n = ; normal cytology, n = ). hpv type was undetermined in one sample. among single infection with ain i, one specimen was infected with a low risk (lr)type (hpv ). nineteen anal swab specimens were identified as hpv positive by fap primers detection (female anal swabs, n = ; male anal swabs, n = ) ( table ). overall α-type strains were detected in samples with fap primers which were not previously detected by my / primers. all but one α-type strains were also detected with α mucosal hpv multiple infection typed with clart hpv clinical array which included at least one hr type. hpv β types (hpv and hpv ) were found in two specimens co-infected with α hpv types; specimens harboured γ hpv types (hpv and hpv ), both associated with type α hpv multiple infections. all fap untyped strains (n = ) were found in anal specimens infected by α types with at least one high hr type. considering cytological aspects, / samples, which resulted positive with fap primer pcr, had ain (i, ii) lesions, while / samples showed normal cytology (table ) . among anal swabs from females (n = ), only samples ( . %), previously tested my / hpv positive, resulted hpv positive with fap primer (hpv , α species; hpv , α species; hpv , α species). no untyped hpv strains were observed among female samples. overall, β genus prevalence was % and γ genus was . % including untyped isolates. mean of cd t cell count was . ± . cells/μl. overall / patients ( . %) were under antiretroviral therapy (art). all patients without art had a cd t cell count > / μl. current art use was not associated with risk of β and ɣ infection (chi square test, p > . ). cervical swabs-twelve specimens showed a single hpv infection, and had multiple infections by clart hpv array; / samples harboured at least hr genotype. cytology findings were available for specimens already resulted positive by clart hpv clinical array: had normal cytology, were asc-us or low grade squamous intraepithelial lesion (lsil), harboured hpv single infections, showed hpv multiple infections, and were high grade squamous intraepithelial lesion (hsil). all my / hpv negative women (n = ) showed normal cytological findings. eleven/ cervical samples resulted fap positive ( . %). however, only hpv types were not previously detected by my / primers. five samples harboured α types (hpv , n = α ; hpv , n = , α ; hpv α , n = ), and only one specimen gave positivity for β (hpv ) ( . %). the mean cd t cell count was ± cells/μl. ninety-six percent of women were receiving art per who guideline of starting art at cd t cell count < cell/mm . the woman harboring β hpv strain was under art. she had a cd t cell count of cells/μl and hiv rna viral load was not detected. overall cervical and anal β and γ type positivity was not related to immune status as measured by cd t cell count (see table ) and art treatment. furthermore, the immune-re constitution could potentially prevent the persistence of β and ɣ types in anal and cervical sites or alternatively, β and ɣ types might show to have a less tropism to these anatomical sites. for the phylogenetic analysis of our untyped hpv samples we used partial cds sampled sequences from major capsid protein l fap pcr products and reference sequences. our reference sequences represent classified and untyped hpv sequences (see table ). according to our phylogenetic analysis, of our sampled sequences (q , q , q , q , q and q ) clustered with previously classified reference sequences from species , , , and -γ genus. sequences q , q , q and q remained unclassified while showing great similarity with unclassified genomic regions af (fams ), kp , and af . respectively (see fig. and table ). in hnc, particularly oropharyngeal cancer, the true prevalence and involvement of hpv in the carcinogenetic process is still unknown. in fact, many studies report low prevalence of hpv obtained with systems used for the determination of hpv in cervical cancer screening (specific for α types), while other authors found higher prevalence when they used a platform able to detect hpv types belonging to genera other than α genus [ ] both in oral cavity and in oral cancer [ , ] . agalliu recently found a correlation between the infection of some β, ɣ hpv types and oscc [ ] . furthermore, a recent meta-analysis provided additional evidence of the involvement of β hpv types in the development of scc in immunocompetent individuals [ ] . additionally, several mechanistic studies have consistently showed that e and e from several β hpv types are able to target cellular proteins, such as p and prb, and to deregulate fundamental events involved in cellular transformation [ ] . thus β and ɣ hpv types detection could be included in a hpv screening test for oral cancer prevention, while a correct and suitable detection of hpv infection is becoming a priority. currently, there are few studies that compare the hpv prevalence obtained using the protocols, which are normally for cervical cancer screening with those obtained using methods that are able to detect hpv types belonging to genera other than α or new ones. in this study, in order to assess the impact of pcr system in hpv prevalence determination, we carried out the detection of hpv with my / primer set, used in several genotyping molecular assays (for example linear array hpv genotyping test, roche molecular system, ca usa; clart hpv clinical arrays, genomica, madrid, spain,) and fap primers able to detect α, β, γ hpv types in several specimens. in oropharyngeal swabs samples, my / primer set was able to detect . % ( / ) of hpv positive samples, while fap set of primers detected the presence of hpv in / samples ( . %). beta hpv was the main genus detected with a prevalence of . % in hpv positive samples. this prevalence was in agreement with bottalico et al. that used fap primer and my / for detecting hpv in oral swabs [ ] . hpv was the most frequent β type and hpv the main type among β species as described by bottalico [ ] . hpv , which was reported as the main β type in forslund study [ ] , was absent in our samples. hr, namely hpv , was observed only in one specimen from an hiv positive subject, whereas hr hpv were frequently observed in bottalico study group [ ] . the low frequency of hr could be imputed to immune status of the patients. our patients were under haart therapy and most of them had a cd t cell count > /μl. no severe compromise of the immune-system could have limited a persistence of hr in this anatomical site. unfortunately, no information was given in the bottalico paper on patient cd t cell count, and this fact did not allow a correct comparison of these results. according to previous data reported by forslund et al. [ ] , no difference in hpv prevalence was observed among men and women suggesting that host specific factors could contribute to hpv persistence and neoplasia development. gamma genus represented . % of hpv positive samples. to note, / γ strains were untyped. phylogenetic analysis revealed that samples (q and q ) belonged to γ species, while q to γ species and q to γ species. interestingly, the oral untyped genotypes fell in different genera and cluster with the anal untyped strains, suggesting a specific tropism of these gamma types for oral mucosa. gamma and were the most representative γ species described by bottalico in oral rinse and agalliu in hnc, suggesting their potential involvement in oscc development. unlike forslund and hampras [ , ] , we did not identified any β , β , β hpv types. this may be due to the types of biological samples that were used or to differences in the efficiency of extraction methods that were applied, which could have influenced the outcome of pcr [ ] . finally, differences in results might also be explained by the geographical distribution of hpv types. in general, differences in hpv β and γ types and their prevalence were observed by the luminex platform. according to him study [ ] , among β genotypes, hpv and hpv were the prevalent genotypes. while, among β strains, hpv was the most represented,and it was never detected in our samples. interestingly hr hpv was also the main β type observed also in moscicki study, whereas hpv represented the main β type [ ] . a different pattern of β types could be related to a different cellular input as described in a previous study [ ] , where several β hpv types showed essentially increasing prevalence with increasing dna input. beta hpv types , , , , , , and showed increased prevalence only in higher dna input groups [ ] . this may impose compromises in comparisons between studies; for example, our hpv negative result could be explained by an insufficient dna input in the pcr assay. further research is needed to establish whether there is an influence between cell number input and hpv β detection in oropharyngeal anatomical site and which cut-off would be suggested to avoid false negative results. possible type-specific differences in sensitivity to cellular input have to be evaluated in wider studies. if they are confirmed, they may be explained by the different sensitivities in detection systems and/or in the fig. a maximum likelihood phylogenetic analysis was inferred on unknown collected samples (bold) and reference sequences using the gtrcat model. out of unknown samples, sequences were clustered within previously classified hpv species lineages. bootstrap supports lower than % were excluded from the tree. genbank accession number: mh -mh different viral load spectra. we detected α strains in anal site fap primers that had not been typed by genomica, and β types, hpv and hpv , that had not been reported among oropharyngeal swabs. Βeta types frequency was sensibly lower to that found by luminex system reported by torres et al. [ ] which found . % of β types in anal swabs from hiv positive msm and . % among hiv negative msm. in this study, hpv and hpv were the prevalent types among β species, while hpv and hpv were the most frequently observed β types. among γ genus, the γ species was the most prevalent in both msm hiv positive and -negative groups, while we observed only γ untyped strains. donà reported higher prevalence of β ( . %) and γ types ( . %) in a group of msm similar to ours using the luminex system [ ] . some hypothesis could put forth to explain these data: i) the luminex system could be much more sensitive than the fap system, or ii) that cross-reactivity could occur in β and γ types detection when α multiple infections are present. in literature, cross-reactivity was also observed among α genotypes. preisler observed cross-reactivity both in lr and hr genotypes using hc , cobas, and aptima assays, despite what manufacturer claims: about % of hpvdna results in primary screening accounting for cross-reactivity, regardless of the assay [ ] . to obtain improved analytical and clinical performance, cross-reactivity studies should be focused, since this aspect could influence the effectiveness in a future head neck hpv cancer screening. in cervical swabs the fap primers mainly showed the presence of α genotypes (hpv , α ; hpv , α ) which were not detected by clart hpv clinical array/ my - primers and β type (hpv ). this low prevalence of β types confirms the data reported by bottalico et al. [ ] , which found a prevalence of % of the β types and % of the γ types in the cervical samples, emphasizing a weak β and γ type tropism towards the female genital mucosa. however, the genotypes hpv and hpv , detected by bottalico in cervical specimens, were never observed among our cervical samples, suggesting a different geographic distribution of hpv β and γ type similarly to that described for α genus. conversely, these findings were in disagreement with those obtained by luminex system, as described in previous study [ ] . a quantitative detection of the viral load in hair follicles demonstrated that the β genotype copy number was considerably lower than that reported for mucosal high-risk types from α genus in cervical tissue [ ] . thus, only comparable viral load detection studies with different methods and the potential for multiple infections detection could explain these differences in prevalence of β types, which are reported in literature. overall, our results confirmed a prevalence of > % for hpv strains in the oropharyngeal anatomical district. the genus β and γ were predominant when the analysis was carried out with fap primers. the discrepancy on the prevalence and hpv types reported in other studies [ ] [ ] [ ] ] seems to be due to the detection system: highest prevalence was always obtained with the luminex system. different sensitivity and specificity of hpv detection methods were also a problem in the determination of α hpv types, as described in a systematic review where approximately - % of all positive results showed discordance [ ] . a global proficiency program like labnet for α types in cervical hpv infection surveillance programs should be planned also for α, β and γ hpv type detection in oropharyngeal samples [ ] . standardizing methods for oral sample collection and hpv detection would ensure comparability between different detection methods in oral cavity samples [ , ] . in addition, considering that γ genus has been growing rapidly (currently γ types have been identified) surpassing α and β genera, and that % of the new types deposited in the hpv center within belonged to γ genus [ - ], our data reinforces the relevance of using primer sets able to detect a wide spectrum of hpv strains including β and γ types as well as new genotypes for hpv detection in the oropharyngeal anatomical site. this seems to be a crucial point since the meta genomic approach applied in some analysis [ ] should be used with caution, taking in account the possibility of having an overestimation of hpv types [ ] and requiring a confirmation of positivity by the sanger method. additional file worldwide burden of cancer attributable to hpv by site, country and hpv type casecontrol study of human papillomavirus and oropharyngeal cancer associations of oral α-, β-, and γ-human papillomavirus types with risk of incident head and neck cancer detection and quantitation of human papillomavirus (hpv) dna in the sera of patients with hpv-associated head and neck squamous cell carcinoma hpv infections and tonsillar carcinoma human papillomavirus (hpv) in head and neck cancer. an association of hpv with squamous cell carcinoma of waldeyer's tonsillar ring incidence of human papillomavirus (hpv) positive tonsillar carcinoma in stockholm, sweden: an epidemic of viral-induced carcinoma? hpv dna, e /e mrna, and p ink a detection in head and neck cancers: a systematic review and meta-analysis preventable exposures associated with human cancers prevalence of microsatellite instability, inactivation of mismatch repair genes, p mutation, and human papillomavirus infection in korean oral cancer patients oral human papillomavirus in healthy individuals: a systematic review of the literature characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals the oral cavity contains abundant known and novel human papillomaviruses from the betapapillomavirus and gammapapillomavirus genera betapapillomaviruses in the anal canal of hiv positive and hiv negative men who have sex with men beta and gamma human papillomaviruses in the anal canal of hiv-infected and uninfected men who have sex with men prevalence of beta and gamma human papillomaviruses in the anal canal of men who have sex with men is influenced by hiv status cervical infection with cutaneous beta and mucosal alpha papillomaviruses prevalence and epidemiologic profile of oral infection with alpha, beta, and gamma papillomaviruses in an asian chinese population the use of polymerase chain reaction amplification for the detection of genital human papillomaviruses identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms a broad range of human papillomavirus types detected with a general pcr method suitable for analysis of cutaneous tumours and normal skin molecular methods for identification and characterization of novel papillomaviruses diversity of human papillomaviruses in skin lesions multiple oral carcinomas associated with a novel papillomavirus in a dog beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants prevalence and risk factors for anal human papillomavirus infection in human immunodeficiency virus (hiv)-positive and high-risk hiv-negative women relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women high diversity of alpha, beta and gamma human papillomaviruses in genital samples from hiv-negative and hiv-positive heterosexual south african men an anal cancer screening program for msm in italy: prevalence of multiple hpv types and vaccine-targeted infections frequency and multiplicity of human papillomavirus infection in hiv- positive women in italy oral human papillomavirus dna detection in hiv-positive men: prevalence, predictors, and co-occurrence at anal site enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models beta-hpv types in patients with head and neck pathology and in healthy subjects association between β-genus human papillomavirus and cutaneous squamous cell carcinoma in immunocompetent individuals-a metaanalysis the biology of beta human papillomaviruses the nasal mucosa contains a large spectrum of human papillomavirus types from the betapapillomavirus and gammapapillomavirus genera prevalence and concordance of cutaneous beta human papillomavirus infection at mucosal and cutaneous sites analysis of the effect of dna purificationon detection of human papillomavirus in oral rinse samples by pcr prevalence and transmission of beta and gamma human papillomavirus in heterosexual couples prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular dna input crossreactivity profiles of hybrid capture ii, cobas, and aptima human papillomavirus assays: split-sample study beta-papillomavirus dna loads in hair follicles of immunocompetent people and organ transplant recipients concordance of beta-papillomavirus across anogenital and oral anatomic sites of men: the him study concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review continuing global improvement in human papillomavirus dna genotyping services: the and hpv labnet international proficiency studies detection of oral hpv infection -comparison of two different specimen collection methods and two hpv detection methods oral human papillomavirus infection incidence and clearance: a systematic review of the literature international standardization and classification of human papillomavirus types towards quality and order in human papillomavirus research hpviewer: sensitive and specific genotyping of human papillomavirus in metagenomic dna we are grateful to all member of virology laboratory inmi l. spallanzani for sample collection. we thank dr. mariana badescu for english editing. the authors declare that they have no competing interests. key: cord- -l ghggu authors: hoang, minh; lin, wei-hao; le, van phan; nga, bui thi to; chiou, ming-tang; lin, chao-nan title: molecular epidemiology of canine parvovirus type in vietnam from november to february date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: l ghggu background: canine parvovirus type (cpv- ) was first identified in the late s; it causes intestinal hemorrhage with severe bloody diarrhea in kennels and dog shelters worldwide. since its emergence, cpv- has been replaced with new genetic variants (cpv- a, cpv- b, and cpv- c). currently, information about the genotype prevalence of cpv- in vietnam is limited. in the present study, we investigated the genotype prevalence and distribution of cpv- in the three regions of vietnam. methods: rectal swabs were collected from dogs with suspected cpv- infection from northern, central, and southern vietnam from november to february . all samples were identified as parvovirus positive by real-time pcr, and further genotyping was performed using a simpleprobe® real-time pcr assay. results: of the vietnamese cpv- isolates, isolates ( . %) were identified as cpv- a, isolates ( . %) were identified as cpv- c and isolates ( . %) were untypable using the simpleprobe® real-time pcr assay. in northern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in central vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in southern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. cpv- b was not observed in this study. the vp genes of cpv- c in vietnam are more genetically similar to those of cpv- c strains in china and taiwan than to those of prototype cpv- c strains (fj ) or the first vietnamese cpv- c (ab ). conclusion: the present study provides evidence that cpv- c is the most prevalent variant in vietnam. phylogenetic analysis demonstrated that the recent vietnamese cpv- c isolates share a common evolutionary origin with asian cpv- c strains. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. canine parvovirus type (cpv- ) is one of the most dangerous enteropathogens, causing fatal disease in dogs and puppies worldwide [ ] . cpv- is a nonenveloped, small dna virus with a diameter of approximately nm and a single-stranded dna genome of approximately kb [ ] . cpv- belongs to the genus parvovirus in the family parvoviridae, which includes feline panleukopenia virus (fpv), mink enteritis virus, raccoon parvovirus, and porcine parvovirus [ ] . clinical manifestations of cpv- infection are characterized by intestinal hemorrhage with severe bloody diarrhea; other clinical signs include anorexia, depression and vomiting [ , , ] . cpv- is a rapidly evolving virus, leading to the mutation of novel variants since its first recognition in the late s [ ] . for example, cpv- a was approximately discovered in in the usa, and cpv- b and c were identified in and in the usa and italy, respectively, based on residue (asn in a, asp in b, and glu in c) in the vp protein of the parvovirus [ , ] . when a novel cpv- variant appears, it very rapidly replaces old variants [ , ] . in recent decades, cpv- c has been found to be widespread in european countries [ , ] , the usa [ ] , south america [ ] [ ] [ ] and africa [ , ] . the first reported occurrence of the cpv- c variant in vietnam was in [ ] ; however, since then, this strain has not been prevalent in asia [ ] . surprisingly, novel asian cpv- c isolates were identified in china [ ] [ ] [ ] [ ] , taiwan [ , ] , laos [ ] and thailand [ ] in the past few years. in vietnam, cpv- infection was first observed as sporadic cases in (unpublished data). subsequently, after its first emergence, there were widespread outbreaks of canine hemorrhagic enteritis with high morbidity and mortality occurring across the whole country [ ] . along with the increasing number of pet dogs in vietnam, cpv- infection has emerged as a veterinary public health concern that greatly affects puppies because of its high mortality and morbidity. however, current information related to the antigenic types of cpv prevailing in vietnam is poorly understood. thus, in the present study, we investigated the genotype prevalence and distribution of cpv- from naturally infected dogs in three regions of vietnam using a simplep-robe® real-time polymerase chain reaction (pcr) assay. rectal swabs were collected from dogs with suspected cpv- infection from northern, central, and southern vietnam from november to february . these dogs had displayed clinical symptoms of cpv- infection, including diarrhea or bloody diarrhea. fecal samples were collected from dogs with alimentary signs (diarrhea or bloody diarrhea) by inserting a swab into the rectum (~ cm), rotating the swab and then resuspending the swab in ml of phosphate-buffered saline. all specimens were transported on ice and stored at - °c at the key laboratory for biotechnology and animal disease, vietnam national university of agriculture, hanoi, vietnam. information including the sampling year, age, clinical history, and cpv- types of the sampled dogs is summarized in additional file . total dna was extracted using a genomic dna mini kit (geneaid biotech, ltd., taipei, taiwan) following the manufacturer's protocol. all of the clinical specimens were confirmed to be infected with parvovirus by real-time pcr, as described by lin et al. [ ] . parvovirus-positive samples were further characterized by a simpleprobe® real-time pcr assay, following the method developed by hoang et al. [ ] . first, the reaction was carried out in a final volume of μl containing mm mgcl , pm simpleprobe® (tib molbiolgmbh, berlin, germany), pm and pm each of primers parvo-f ( ′-aca cct gag aga ttt aca tat ata gca ca- ′) and parvo-a ( ′-att agt ata gtt aat tcc tgt ttt acc tcc- ′) , μl x light cycler genotyping master mix and μl x diluted template dna. real-time pcr was cycled under the same thermal conditions as those previously described for the simpleprobe® assay [ ] . to clone full length vp , we performed pcr amplification using the primer pair vp f ( ′-cggtgcagg acaagtaaaa - ′)/vp r ( ′-ggtgctagttgata tgtaata - ′) that amplified a bp fragment of the gene encoding the capsid protein. the pcr conditions were as follows: denaturation at °c for min; cycles of denaturation at °c for s, annealing at °c for min, and extension at °c for min; and a final extension step at °c for min. pcr products were electrophoresed, followed by the purification of electrophoresis products by a wizard® sv gel and pcr clean-up system (promega corporation, usa). the pcr products obtained from the previous step were cloned by a t&a® cloning kit before being sent for automated sequencing (mb mission biotech, inc., taiwan). the resulting sequences were compared with reference fpv (m ), cpv- (m ), cpv- a (m , and m ), new cpv- a (ay , ab , eu , jq , kf , and kr ), cpv- b (m and m ), new cpv- b (ab , ab , ab , ab , ab , ab , ab , ay , ay , jq , kr , and kr ), cpv- c (fj , ab , fj , km , kr , kt , kt , ky , mf , and ku ), cpv- a vaccine strain (fj ) and previous vietnamese strains (ab , ab , ab , ab , ab , ab , ab ). the clustal w method and megalign program (dnastar, madison, wi, usa) were used for multiple alignments of the nucleic acid and amino acid sequences. phylogenetic analyses were conducted by the maximum likelihood methods using mega based on the tamura-nei model [ ] . a total of specimens were collected from suspected parvovirus-infected dogs in three regions of vietnam, including northern, central and southern vietnam. all samples were positive for parvovirus dna. the age of the sampled dogs ranged from month to months old. the demographic summary of puppies that tested positive for parvovirus by real-time pcr is also presented in additional file . the number of puppies less than months old accounted for . % ( / ) of the sample, dogs of to months of age accounted for . % ( / ) of the sample, and dogs older than months accounted for . % ( / ) of the sample. for samples, the age group was unspecified. in terms of gender, . % ( / ) of the sample was male dogs, and . % ( / ) was female dogs. for samples, the gender was not reported. in the northern areas of vietnam, cpv- cases often peak in november and january, while the peak time for central areas is from december to march, and the peak time for southern areas is in june and july (fig. ). among the samples collected from the three regions in vietnam, samples ( . %) were clearly genotyped according to melting temperature analysis using a simpleprobe® real-time pcr assay (table ) . however, there were three cases ( . %) from northern vietnam for which differentiation was not possible by the simpleprobe® real-time pcr assay (additional file ). among samples, six isolates ( . %) were identified as cpv- a and isolates ( . %) were identified as cpv- c. in northern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in central vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively. in southern vietnam, the percentages of cpv- a and cpv- c were . % ( / ) and . % ( / ), respectively (fig. ) . cpv- b was not observed in this study (fig. ) . our results showed that cpv- c is currently the most prevalent cpv- field strain circulating in vietnam. a total of dogs ( . %) were confirmed to be vaccinated against cpv- . among cpv- vaccinated dogs, were younger than months old, and were months old or older; the age of the remaining dogs could not be accurately determined. interestingly, all vaccinated dogs were identified as having cpv- c infection using the simpleprobe® real-time pcr assay. to investigate the association and diversity of cpv- strains circulating in vietnam, we sequenced and analyzed complete vp genes of cpv- strains collected from the three regions of vietnam. phylogenetic analysis based on the nucleotide sequences of the complete vp gene revealed two clusters that were prototype cpv- c strains and asian cpv- c clusters ( (fig. ) . the vp genes of cpv- c in vietnam are more genetically similar to cpv- c strains in china and taiwan than to prototype cpv- c strains (fj ) or first vietnamese cpv- c (ab ) (fig. ). in addition, one vietnamese strain (dn ) in this study was clustered with a cpv- a vaccine strain (fj ) (fig. ). there were several nucleotide mutations in the vp gene of all strains (table ), but only mutations (nucleotide position , , , , , and ) table . for the complete vp analysis, one sequence with an asn and sequences with a glu at position were classified as cpv- a and cpv- c, respectively. all cpv- c strains showed substitution at position ala gly, phe tyr, tyr ile, and gln arg ( table ) . thirteen of the cpv- c strains obtained showed a unique substitution at position (ile to met) caused by mutation of tat to tgt at nucleotide positions - of the vp gene (table ). cpv- c is currently the most prevalent genotype in asian countries [ ] [ ] [ ] [ ] [ ] [ ] . the first asian cpv- c case was detected in vietnam in [ ] . however, information regarding the current genotype prevalence of cpv- strains in vietnam is limited. this study is the first to investigate the genotype prevalence of cpv- in regions of vietnam. similar to other asian countries [ ] [ ] [ ] [ ] [ ] [ ] , the cpv- c variant appears to be the most prevalent genotype in the dog population in all regions of vietnam. surprisingly, no cpv- b cases were found in vietnam in the present study; however, according to previous reports, cpv- b had the highest detection rate in vietnam in [ ] ( table ). the reasons for this discrepancy may be as follows: i) current cpv- b vaccines are effective in preventing and controlling the disease in vietnam; or ii) there is limited or no protection from current vaccines against the cpv- c variant, and this variant rapidly replaced the previous circulation of cpv- strains. as such, we used a sybr green-based real-time pcr assay, which was sensitive, specific and reliable for the detection of cpv- , fpv and porcine parvovirus dna [ ] . however, there were three parvovirus-positive cases from northern vietnam for which differentiation was not possible by our simplep-robe® real-time pcr assay. this issue may be due to i) unusual changes in the probe sequence region [ ] ; or ii) the infection of vietnamese dogs by other parvovirus species for which the sequence analysis has not been investigated. the country of vietnam is divided into three regions with different climates. northern vietnam has a humid subtropical climate, while the central region has a tropical monsoon climate, and southern vietnam has a tropical savanna climate. another factor that may affect the study is the humidity in vietnam, which is - % on average. however, because of differences in latitude and topography, the climate tends to be markedly different across regions, which may cause the difference in the timing of parvovirus outbreaks in specific regions. in the northern part of the country, due to more seasons than other regions ( seasons in total), the seasonal periods of parvovirus outbreaks are usually from october to november (winter transition time), from december to january (spring transition time), and from march to may, with a peak in april (summer transition time) (fig. ) . northern areas show a disease period spread over many months, whereas outbreaks often occur at a more concrete, specific time in the central and southern parts of the country. in fact, in central vietnam, the period from december to march is the time when parvovirus infection increased rapidly in dogs. in the southern part of the country, small dogs are more likely to be affected during the period from june to august when the weather is beginning to switch from the dry to rainy season. thus, cpv- disease can occur in vietnam at any time of the year depending on the geographic location; however, when there is a change in climate, temperature and humidity make puppies susceptible to infection and cause cpv- to become widespread in the environment due to prolonged survival time. various contradicting studies emphasize the disease's seasonality in different specific geographical regions. some scientists have indicated that the highest incidence of this disease is detected in spring and summer [ , ] , while the opposite is true for other locations [ ] [ ] [ ] [ ] . dogs aged months to months accounted for the highest detection rate ( / ; . %), while this rate for the month to month age group was . % ( / ), and the rate for dogs over months old was only . % ( / ). for samples, the age group was not reported. the percentage of dogs under months of age who were greatly affected has been previously reported [ , ] . the reason dogs from months to months old are easily susceptible to cpv is due to the decrease in maternal antibodies [ , ] . in addition, the higher incidence of cpv- in dogs younger than months old might be due to quicker intestinal crypt cell multiplication with a higher mitotic index by changes in bacterial flora and daily diet during weaning time [ , ] . very few cases are recorded over year of age; the cause may be slight exposure to the virus, leading to antibody production in the host; previous vaccination of animals; or another reason not yet identified. dogs in the to month age group still exhibit cpv disease, which may be because of poor vaccine preservation or incorrect vaccination timing [ ] . we found that the proportion of infected females was less than that of males. of positive cases, were females ( . %), and ( . %) were males. coincidently, the current finding is consistent with studies by thomas et al. [ ] and gombac et al. [ ] , as well as other reliable publications [ , , [ ] [ ] [ ] . many assumptions could be analyzed; however, the most relevant cause could be that the majority of samples were collections from male dogs during the study. another explanation for this result could be that male dogs are more likely to be infected; in fact, more male dogs were admitted for treatment than female dogs. however, there is reportedly no influence of sex on the incidence of cpv [ , ] . the high incidence in male dogs may also be due to some behaviors and habits of pet owners or the hobby of selecting and keeping male dogs [ ] . a large number of dogs have been vaccinated against cpv- but still suffer from the disease, with most of the cases ( / ; . %) months of age and older. this issue may be related to failure to properly use the vaccine or to preserve the vaccine [ , ] . many studies have shown that cpv- and cpv- b vaccines provide protection against the cpv- a/ b virus and provide complete protection against the new cpv- c variant in dogs for up to years [ ] [ ] [ ] . however, there are some disagreements regarding the use of traditional vaccines against the new antigen variant cpv- c [ , ] . similar to a study conducted in taiwan, four of cpv- c-infected dogs died despite vaccination, including one adult dog that completed the vaccination program [ ] . this phenomenon has also been seen in vietnam where % of vaccinated positive cases were of genotype c. many cpv- vaccines are being used in vietnam; however, there is little documentation of the effects of cpv- vaccination against the current cpv- c variant. therefore, the efficacy of the current vaccine against the cpv- c variant remains to be evaluated. currently, there are many publications regarding the circulation of the cpv- c strain in many countries in asia [ - , - , , ] although the first publication on cpv- c was in vietnam in [ ] , the cpv- c variant has not been prevalent in asia. surprisingly, novel asian cpv- c isolates were reported in china [ ] [ ] [ ] [ ] , taiwan [ , ] , laos [ ] , and thailand [ ] . this finding suggests that novel cpv- c is now becoming increasingly prevalent in asian countries. based on the results of the phylogenetic analysis, the cause for the large amount of cpv- c detected in vietnam is the invasion of a foreign strain from to . in addition, there was no phylogenetical relation between vietnamese cpv- c and prototype cpv- c strains (european or american strains) or the past vietnamese cpv- c in , while these strains are similar to the recent asian cpv- c strains (fig. ) . phylogenetic analysis revealed that the recent vietnamese cpv- c strains share a common evolutionary origin with asian cpv- c strains. this finding could be explained by dog imports from china and other neighboring nations with a lack of oversight due to a long shared border with these countries and the effort needed from authorities to apply restrictions. amino acid substitutions of ala gly, phe tyr, tyr ile and gln arg of cpv- c have been observed in china [ , , ] and taiwan [ , ] . however, the functions of cpv- residues , , and are still unknown and remain to be elucidated. the substitution of ile met is unique to the vietnamese cpv- c strains. residue is not exposed on the capsid surface [ ] , and substitutions in this position may not affect the antigenicity of the virus. however, agbandie's study showed that the binding of dna within the internal surface of the parvovirus protein shell may cause specific conformational changes in the protein [ ] . therefore, the function of residue remains to be elucidated. this study is the first cpv- epidemiological survey in vietnam to document the presence and relative distribution of cpv- variants in three regions of vietnam over the past few years. phylogenetic analysis demonstrated that the recent vietnamese cpv- c isolates share a common evolutionary origin with asian cpv- c strains. it is important for veterinarians and dog owners to understand the current geno-prevalence of cpv- and effective prevention methods based on outbreak periods. availability of data and materials not applicable. canine parvovirus: a review of epidemiological and diagnostic aspects, with emphasis on type c nucleotide sequence and genome organization of canine parvovirus the emergence of parvoviruses of carnivores rapid antigenic-type replacement and dna sequence evolution of canine parvovirus isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis natural variation of canine parvovirus evidence for evolution of canine parvovirus type in italy a canine parvovirus mutant is spreading in italy characterisation of the canine parvovirus type variants using minor groove binder probe technology occurrence of canine parvovirus type c in the united states first detection of canine parvovirus type c in south america evolution of canine parvovirus in argentina between years and : cpv c has become the predominant variant affecting the domestic dog population genotyping of canine parvovirus in western mexico molecular epidemiology of canine parvovirus in morocco molecular characterization of canine parvovirus- variants circulating in tunisia a novel antigenic variant of canine parvovirus from a vietnamese dog identification of a novel canine parvovirus type c in taiwan co-circulation of the rare cpv- c with unique gln arg substitution, new cpv- b with unique thr ala substitution, and new cpv- a with high prevalence and variation in heilongjiang province typing of canine parvovirus strains circulating in north-east china typing of canine parvovirus strains circulating in north-east china continuing evolution of canine parvovirus in china: isolation of novel variants with an ala gly mutation in the vp protein phylodynamic and genetic diversity of canine parvovirus type c in taiwan molecular characterization of canine parvovirus in vientiane emergence of canine parvovirus type c in domestic dogs and cats from thailand development of sybr green-based real-time pcr for the detection of canine, feline and porcine parvoviruses a simpleprobe® real-time pcr assay for differentiating the canine parvovirus type genotypereal-time pcr assay for differentiating the canine parvovirus type genotype mega : molecular evolutionary genetics analysis version . canine parvovirus in new zealand: epidemiological features and diagnostic methods risk factors associated with parvovirus enteritis in dogs: cases ( - ) canine parvovirus in a commercial kennel: epidemiologic and pathologic findings aspects of the diagnosis, pathogenesis and epidemiology of canine parvovirus parvovirus enteritis in dogs based on autopsy statistics - clinical and epidemiological aspects of canine parvovirus (cpv) enteritis in the state of molecular epidemiology of canine parvovirus in southern india incidence of canine parvovirus in diarrhoeic dogs by polymerase chain reaction frequency of cpv infection in vaccinated puppies that attended puppy socialization classes a modified live canine parvovirus vaccine. ii. immune response polymerase chain reaction based epidemiological investigation of canine parvoviral disease in dogs at bareilly region retrospective study of canine parvovirosis in slovenia. case report prevalence of canine parvovirus infection in assam occurrence of canine parvovirus in dogs from henan province of china in - dog ecology and demography information to support the planning of rabies control in machakos district epidemiological study of canine parvovirus epizootiological status of canine viral haemorrhagic gastroenteritis in bhubaneswar city epidemiology of parvovirus and coronavirus infections in dogs in assam evolution of canine parvovirus-a need for new vaccines? do two current canine parvovirus type and b vaccines provide protection against the new type c variant? vaccination with canine parvovirus type (cpv- ) protects against challenge with virulent cpv- b and cpv- c canine parvovirus type vaccine protects against virulent challenge with type c virus are licensed canine parvovirus (cpv and cpv b) vaccines able to elicit protection against cpv c subtype in puppies?: a systematic review of controlled clinical trials canine parvovirus: the worldwide occurrence of antigenic variants occurrence of canine parvovirus type c in the dogs with haemorrhagic enteritis in india the first detection of canine parvovirus type c in china structure determination of feline panleukopenia virus empty particles authors' contributions mh and whl performed the molecular data analysis and wrote the manuscript. bttn performed the cloning and sequence analysis. vpl and mtc managed the study, provided materials and reagents, contributed to the interpretation of the data, and cowrote the manuscript. cnl designed and analyzed the experimental data and wrote the manuscript. all of the authors read and approved the final manuscript. the study did not involve any animal experiments. the institutional animal care and use committee (iacuc) of vietnam national university of agriculture did not deem it necessary for this research group to obtain formal approval to conduct this study. not applicable. the authors declare that they have no competing interests. key: cord- -yol hid authors: xu, tian-min; lin, bin; chen, cong; liu, long-gen; xue, yuan title: non-optimal effectiveness of convalescent plasma transfusion and hydroxychloroquine in treating covid- : a case report date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: yol hid background: convalescent plasma (cp) transfusion was reported to be effective in treating critically ill patients with covid- , and hydroxychloroquine could potently inhibit sars-cov- in vitro. herein, we reported a case receiving combination therapy with cp transfusion and hydroxychloroquine for the first time. case presentation: laboratory findings showed high lactic acid level ( . mmol/l) and c-reactive protein (crp, . mg/l), and low white blood cell count ( . × ( )/l) in a -year-old chinese man, who was diagnosed with severe covid- . cp was intravenously given twice, and hydroxychloroquine was orally administrated for a week ( . g, three times a day). the lactic acid and c-reactive protein levels remained high ( . mmol/l and . mg/l, respectively), while the arterial oxyhemoglobin saturation decreased to % with a low oxygenation index (oi, mmhg) on day after cp transfusion. his temperature returned to normal and the oi ascended above on day . moreover, the rna test remained positive in throat swab, and computed tomography revealed severe pulmonary lesions on day after admission. conclusion: these findings suggested that the effectiveness of combination therapy with cp and hydroxychloroquine may be non-optimal, and specific therapy needs to be explored. coronavirus disease (covid- ), caused by severe acute respiratory syndrome coronavirus (sars-cov- ), is a pandemic that is rapidly spreading worldwide. it has been reported that - % of severe patients with mild atypical symptoms initially, can rapidly progress to acute respiratory distress syndrome, the main cause of respiratory failure [ ] . few studies have reported the effectiveness of convalescent plasma (cp) transfusion in treating critically ill patients with covid- [ ] [ ] [ ] . the clinical symptoms were significantly improved within days, and the viral load was undetectable within days after cp transfusion [ ] . these results are encouraging and worthy of further investigation. nevertheless, zeng et al. [ ] reported that sars-cov- became undetectable after cp transfusion, but it could not reduce mortality in critically ill covid- patients. moreover, hydroxychloroquine was found to be active against sars-cov- in vitro, and is a treatment option in clinical practice. yao et al. [ ] reported that hydroxychloroquine was more potent than chloroquine to inhibit sars-cov- in vitro. herein, we administered cp transfusion and hydroxychloroquine to a patient with severe covid- , and analyzed their clinical symptoms, oxygenation index (oi), and dynamics of viral load. a -year-old chinese man, who was diagnosed with laboratory-confirmed covid- , was admitted to our isolation ward on march . a week before his admission, he flew in from brazil airport, and was subsequently under home isolation. on march , he presented with fever ( . °c), chills and myalgia. viral rna test from throat swab was positive. the cycle threshold (ct) values of open reading frame ab (orf ab) and nucleocapsid (n) genes by rt-pcr assay were and , indicating a high viral load. computed tomography (ct) scan showed bilateral pneumonia. laboratory findings showed high lactic acid level ( . mmol/l) and c-reactive protein (crp, . mg/l), and low white blood cell count ( . × /l), indicative of severe covid- . oxygen and atomized inhalation of recombinant human interferon-ɑ b was given at admission. cp from two convalescent patients was intravenously given, and hydroxychloroquine (shanghai zhongxi pharmaceuticals, shanghai, china) was orally administrated for a week ( . g, three times a day). as shown in fig. , on day after cp transfusion, the lactic acid and crp levels remained high ( . mmol/l and . mg/l, respectively). the arterial oxyhemoglobin saturation (sao ) decreased to %, while the oi decreased to mmhg, and mechanical ventilation was administered. his temperature returned to normal and the oi ascended above on day , after which the ventilator was withdrawn. on day after cp transfusion, the rna test remained positive in throat swab, and ct revealed severe pulmonary lesions (fig. ) . in addition, no apparent side-effects were found during cp transfusion and hydroxychloroquine treatments. currently, the pathogenesis of covid- remains unclear, and no specific treatment is available. cytokine storm and excessive inflammation coexist and may lead to multiple organ failure. both antiviral therapy and treatment of the systematic response are important for patients with rapid deterioration of covid- . in a study with small sample size, the median time from onset of illness to cp transfusion was . days, and the disappearance of viremia was observed in days after cp transfusion [ ] . the results suggested that cp therapy could potentially improve the clinical outcomes by neutralizing viremia in severe covid- [ ] . in another study, cp transfusion was given at a median of . days after first detection of sars-cov- , the viral load became negative days after cp transfusion, though of patients died eventually [ ] . unexpectedly, the viral load remained detectable on day after combination therapy with cp and hydroxychloroquine. considering the time from onset of illness to cp transfusion in previous studied [ , ] , it is difficult to prove whether the viremia is cured by the cp and antiviral agent, or is a natural course of covid- . indeed, there is a limitation that antibodies against sars-cov- in cp from the two convalescent patients and the present patient are not detected. based on our clinical experience, sufficient evidence supporting the use of cp and hydroxychloroquine in treating covid- is lacking. our findings suggested that the effectiveness of combination therapy with cp and hydroxychloroquine may be non-optimal, and specific therapy needs to be explored. epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study treatment of critically ill patients with covid- with convalescent plasma effectiveness of convalescent plasma therapy in severe covid- patients treatment with convalescent plasma for critically ill patients with severe acute respiratory syndrome coronavirus infection effect of convalescent plasma therapy on viral shedding and survival in covid- patients in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. tian-min xu, bin lin, cong chen, long-gen liu, yuan xue collected and confirmed the data. yuan xue, tianmin xu and bin lin performed the analysis and drafted the manuscript. all authors read and approved the final manuscript. tianmin xu and bin lin contributed equally to this work. derived data are available on reasonable request. this study was approved by the ethics committee of the third people's hospital of changzhou, and was performed in accordance with the declaration of helsinki. written consent was obtained from the patient for publication. all authors declare no conflict of interest. key: cord- - cuk cn authors: plyusnina, angelina; plyusnin, alexander title: recombinant tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: cuk cn tula hantavirus carrying recombinant s rna segment (rectulv) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. our data showed that the lower competitiveness of rectulv could not be increased by pre-passaging in the cell culture. nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. the observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature. recombination in rna viruses serves two main purposes: (i) it generates and spreads advantageous genetic combinations; and (ii) it counters the deleterious effect of mutations that, due to the low fidelity of viral rna polymerases and lack of proofreading, occur with high frequency [ ] . the purging function is, naturally, attributed to the homologous recombination (hrec), i.e. recombination between homologous parental molecules through crossover at homologous sites. hrec was first described for the positive-sense rna viruses [ , ] and subsequent studies lead to the widely accepted copychoice model [ ] . hrec was later shown to occur in rotaviruses thus adding double-stranded rna viruses to the list of viruses capable of recombination [ ] . negativesense rna viruses that occupy the largest domain in the virus kingdom until recently were known to undergo nonhomologous recombination only, forming either defective genomes, like polymerase "mosaics" of influenza a virus di-particles [ ] and "copy-backs" of parainfluenza virus [ ] or hybrids between viral and cellular genes [ ] or between different viral genes [ ] . the first evidence for hrec in a negative-sense rna virus has been obtained on hantaviruses [ , ] . hantaviruses (genus hantavirus, family bunyaviridae) have a tripartite genome comprising the l segment encoding the rna-polymerase, the m segment encoding two external glycoproteins, and the s segment encoding the nucleocapsid (n) protein [ ] . hantaviruses are maintained in nature in persistently infected rodents, each hantavirus type being predominantly associated with a distinct rodent host species [ ] . when transmitted to humans, some hantaviruses cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome, whereas other hantaviruses are apathogenic [ , ] . persistent infection in natural hosts allows for the simultaneous presence of more than one genetically distinct hantavirus variant in the same rodent. this may result in hantavirus genome reassortment [ , ] or recombination, as proposed in the above-mentioned study of sibold et al [ ] who showed a mosaic-like structure of the s rna segment and the n protein of tula hantavirus (tulv). most recently, we have shown transfection-mediated rescue of tulv with recombinant s segment, in which nt - originate from the cell culture isolate moravia/ma v/ (or tulv , for short) [ ] , nt - originate from the strain tula/ma / [ ] , and nt - , that are identical in both variants, can be of either origin. both m and l segments of the recombinant virus (rectulv) originate from tulv [ ] . rectulv was functionally competent but less competitive than tulv . one reason for the observed lower fitness of the rectulv might be that it was generated in the presence of the wt variant, with which it has to compete, and thus not given enough time to to establish a well balanced, mature quasi-species population. we, therefore, decided to compare fitness of tulv with that of rectulv that underwent several passages in cell culture. first, we designed rt-pcr primers able to discriminate between non-recombinant (v-type) and recombinant (rec-type) types of tulv s rna. the resullts presented in fig. show that the primer pairs designed to generate the bp-long products from either v-type or rec-type s rna amplified, indeed, homologous sequences only, whether these were taken along (lines and ) or mixed with the heterologous sequences (lines and ). using the two specific rt-pcr conditions, the presence of v-type and rec-type s rna was monitored on ten sequential passages of the mixture of tulv and rectulv variants (fig. ). s rna of v-type was seen on all passages ( fig. a , lines - ). in contrast, s rna of rec-type, was detected up to the fifth passage (fig. b , lines - ), and then disappeared (fig. b , lines - ). an alternative approach to check the presence of the two different types of s rna using specific primer pairs at the stage of nested pcr gave exactly the same result. the v-type s rna was detected during all ten passages while the rec-type totally disappeared after the th passage (data not shown). these data confirmed our earlier observation [ ] that the transfection-mediated hrec yields functionally competent and stable virus, rectulv. the purified and pre-passaged recombinant virus, however, presented no real match to the original cell adapted variant, tul , it terms of fitness. taking into account that the in situ formed recombinant s rna disappeared from the mixture after four passages [ ] , one should conclude that the lower competitiveness of the recombinant virus seen earlier did not result from its "immature" status. when, under similar experimental settings, tul has been passaging in the presence of another isolate, tulv/lodz, none of the two viruses was able to establish a dominance during ten consecutive passages (plyusnin et al., unpublished data). although relatively short, the observed survival time of the rectulv in the presence of the original variant tul seems to be sufficient for transmission of a recombinant virus, in a hypothetical in vivo situation, from one rodent to another. if transmission is performed in a samplinglike fashion -and this seems to be the case for hantaviruses [ ] -the recombinant would have fair chances to survive. the existence of wt recombinant strains of tulv [ ] supports this way of reasoning. evidence for the recombination in the hantavirus evolution continues to accumulate [ , ] . the genetic swarm of s rna molecules from the rectulv is represented almost exclusively by the variant with a single break point located between nt and nt . the proportion of the dominant variant is larger in the passaged rectulv ( of cdna clones analyzed, or %) than in the freshly formed mixture of recs rnas ( of cdna clones, or %) [ ] . thus, rectulv already represents a product of a micro-evolutionary play, in which the best-fit variant has been selected from the initial mixture of recs rna. whether this resulted from higher frequency of recombination through the "hot-spot" located between nt and nt or from the swift elimination of all other products of random recombination due to their lower fitness (the situation reported for polio-and coronaviruses [ , ] ), or both, remains unclear. we favor the first explanation as the modeling of the s rna folding suggests formation of a relatively long hairpin-like structure within the recombination "hot-spot" (fig. ) . secondary structure elements of this kind, which might present obstacles for sliding of the viral rna polymerase along the template, were suggested as promoters for the template- switching in the early studies on polioviruses [ ] and considered a crucial prerequisite for recombination [ , ] . the hairpin in tulv plus-sense s rna (fig. ) is formed by the almost perfect inverted repeat that includes nt to . in the minus-sense rna, the structure is slightly weaker due to the fact that two non-canonical g:u base pairs presented in the plus-sense rna occur as nonpairing c/a bases in the minus-sense rna. interestingly, in puumala hantavirus, a hairpin-like structure formed by a highly conserved inverted repeat in the '-noncoding region of the s segment seems to be involved in recombi-nation events, leading, however, to the deletion of the hairpin-forming sequences (a. plyusnin, unpublished observations). the role of rna folding in hantavirus recombination awaits further investigation. the data presented in this paper show that the rectulv presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture. the observed survival time of the rectulv in the presence of the monitoring of wt and recs-rna during sequential passages of the mixture of tul and rectulv rectulv (clone ) was purified from the mixture it formed with the original variant, tulv , using two consequent passages under terminal dilutions [ ] . after the purification, rectulv underwent three more passages, performed under standard conditions, i.e. without dilution. the presence of recs-rna on the passages was monitored by rt-pcr and the isolate appeared to have a stable genotype (data not shown). rectulv formed foci similar in size to those of the original variant and grew to the titers × - ffu/ml. vero e cells ( × cells) were infected with the : mixture of rectulv and tulv , approximately ffu alto-gether. after - days the supernatant (~ ml) was collected and rna was extracted from the cells with tripure™ isolation reagent, boehringer mannheim. aliquots ( ml) of the supernatant were used to infect fresh cells; the rest was kept at - °c. the following nine passages were performed in the same way. rt was performed with mulv reverse transcriptase (new england biolabs); for pcr, amplitaq dna polymerase (perkin elmer, roche molecular systems) was used. to monitor the presence of tulv s rna on passages, rt-pcr was performed with primers vf ( 'gcctgaaaagattgaggagttcc '; nt - ) and vr ( 'ttcacgtcctaaaaggtaagcatca '; nt - ). to monitor the presence of rectulv s rna, rt-pcr was performed with primers recf ( 'gccagagaagattgaggcatttc '; nt - ) and hairpin-like structures predicted for the recombination "hot-spot" in the plus-and minus-sense s rna of tulv a g-c a-u ccuuuac cgguuca recr ( 'ttctctcccaattaggtaagcatca '; nt - ). all four primers were perfect matches to the homologous sequences; to the heterologous sequences, the forward primers have five mismatches while the reverse primers have six. alternatively, complete s segment sequences of both variants of tulv were amplified using a single universal primer [ ] and then either of the two pairs of primers was used in nested pcr. authenticity of the pcr amplicons was confirmed by direct sequencing using the abi prism dye terminator sequencing kit (perkin elmer applied biosystems division). evolutionary aspects of recombination in rna viruses genetic recombination with newcastle disease virus, polioviruses and influenza virus. cold spring harbor symp quant biol genetic recombination with poliovirus type : studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain on the nature of poliovirus genetic recombinants intragenic recombination in rotaviruses defective interfering rnas of influenza viruses: origin, structure, expression and interference philadelphia: lippincott-raven publishers increased viral pathogenicity after insertion of a s ribosomal rna sequence into the hemagglutinin gene of influenza virus nonhomologous recombination between the hemagglutinin gene and the nucleoprotein gene of an influenza virus recombination in tula hantavirus evolution: analysis of genetic lineages from slovakia transfection-mediated generation of functionally competent tula hantavirus with recombinant s rna segment family bunyaviridae virus evolution and genetic diversity of hantaviruses and their rodent hosts hantavirus pulmonary syndrome and newly described hantaviruses in the united states molecular epidemiology of hantavirus infections st: naturally occurring sin nombre virus genetic reassortants complete nucleotide sequences of the m and s segments of two hantavirus isolates from california: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome isolation and characterization of tula virus: a distinct serotype in genus hantavirus, family bunyaviridae tula virus: a newly detected hantavirus carried by european common voles molecular evolution of puumala hantavirus phylogenetic analysis reveals a low rate of homologous recombination in negative-sense rna viruses random nature of coronavirus rna recombination in the absence of selection pressure poliovirus rna recombination: mechanistic studies in the absence if selection studies on the reecombination between rna genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants new insights into the mechanisms of rna recombination mechanisms of retroviral recombination the authors thank prof. Åke lundkvist for fruitful discussion and prof. antti vaheri for general support. this work was supported by the research grants rfa and from the academy of finland. the author(s) declare that they have no competing interests. angp participated in the design of the study, carried out the experiments and helped to draft the manuscript. alexp participated in the design of the study and drafted the manuscript. both authors read and approved the final manuscript. key: cord- -cdg im h authors: van beurden, steven j.; berends, alinda j.; krämer-kühl, annika; spekreijse, dieuwertje; chénard, gilles; philipp, hans-christian; mundt, egbert; rottier, peter j. m.; verheije, m. hélène title: a reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted rna recombination date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: cdg im h background: avian coronavirus infectious bronchitis virus (ibv) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. major advances in the study of the molecular biology of ibv have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, ibv strain beaudette. however, most ibv strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. methods: we established a reverse genetics system for the ibv strain h , based on targeted rna recombination in a two-step process. first, a genomic and a chimeric synthetic, modified ibv rna were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) ibv intermediate (mibv). herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (mhv), allowing for the selection and propagation of recombinant mibv in murine cells. in the second step, mibv was used as the recipient. to this end a recombination with synthetic rna comprising the ′-end of the ibv genome was performed by introducing the complete ibv spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. results: targeted rna recombination allowed for the modification of the ′-end of the ibv genome, encoding all structural and accessory genes. a wild-type recombinant ibv was constructed, containing several synonymous marker mutations. the in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental ibv strain h . conclusions: targeted rna recombination allows for the generation of recombinant ibv strains that are not able to infect and propagate in continuous cell lines. the ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated ibv vaccines and for studies into the biology of ibv in general. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. avian coronavirus infectious bronchitis virus (ibv) primarily infects the upper respiratory epithelium of chickens, causing a respiratory disease that is frequently complicated by secondary bacterial pathogens [ ] . in addition, some ibv strains affect the renal tubuli, oviduct and parts of the gastrointestinal tract, leading to pathological lesions in these organ systems, with subsequent reduced weight gain and a drop in egg production. the virus has a worldwide presence in both commercial and backyard chickens, appearing in a wide variety of geno-, sero-and protectotypes [ ] . ibv is currently regarded as one of the economically most relevant viral pathogens in the poultry industry. infectious bronchitis virus is the prototype gammacoronavirus in the family coronaviridae, order nidovirales [ ] . the enveloped virus particles have a positive-sense rna genome of . kb (fig. a ) [ ] . the ′ two-third of the viral genome comprises gene , divided into two large open reading frames a and b, which code for nonstructural proteins primarily involved in rna replication and transcription. the ′ one-third of the viral genome codes for structural proteins: spike protein (s, encoded by gene ), envelope protein (e, encoded by gene c), and membrane protein (m, encoded by gene ), each located in the viral envelope. the nucleocapsid protein (n, encoded by gene ) occurs in the ribonucleoprotein core [ ] . interspersed between the structural genes, coronaviruses carry a variable number of genus specific accessory genes [ ] . most of their gene products are nonstructural, and their expression is not essential for virus replication in vitro [ ] [ ] [ ] [ ] [ ] [ ] . the ibv genome contains the accessory genes and , encoding the proteins a and b, and proteins a and b, respectively [ ] . in addition, an open reading frame located in the intergenic region was identified between genes and [ ] . the typical coronavirus spikes are formed by trimers of the type membrane protein s, which is often proteolytically cleaved into two subunits, s and s [ , ] . the glycosylated s domain forms the 'head' of the spike and contains the receptor-binding domain [ ] . avian gammacoronaviruses typically interact with glycans on the host cell surface. ibv in particular requires α , -linked sialic acids for attachment and entry [ ] [ ] [ ] . the s domain builds the remaining part of the ectodomain (the 'stalk'), the transmembrane domain and the internally located endodomain. the s protein is the main determinant of the coronaviral host species tropism [ ] . many mammalian coronaviruses of the genera alphacoronavirus and betacoronaviruses can be propagated in cultured cells, unlike most avian coronaviruses of the genus gammacoronavirus. ibv can, however, readily be propagated in, and isolated from, embryonated fowl eggs. during passaging in embryonated eggs adaptation occurs, often leading to attenuation. for example, ibv strain h represents the nd serial passage of a massachusetts-like ibv strain isolated in the netherlands [ ] , which causes embryonic death within h post infection (hpi), and still has a residual virulence in young chickens. another passages resulted in the ibv strain h , which is more attenuated and has a lower pathogenicity in young chicks. similar serial passaging of another ibv strain of the massachusetts serotype isolated in the usa resulted in the generation of the non-pathogenic and cell-culture adapted ibv strain beaudette [ ] . in order to study its characteristics, several research groups have independently developed a reverse genetics system (rgs) for ibv which allow the manipulation of its genome [ ] [ ] [ ] [ ] [ ] . all these systems are based on the nonpathogenic cell-culture adapted ibv strain beaudette, or the highly attenuated ibv vaccine strain h . a major drawback of the use of the non-virulent ibv strains beaudette and h [ , ] is the inability to provide insights in the infection process in chickens, as these strains no longer cause a clinically relevant phenotype in vivo. yet, the rgs has provided significant insight in the fundamentals of avian gammacoronavirus replication. key findings include that ibv cell tropism is determined by the spike gene [ , ] , that the low virulence of ibv beaudette is caused by changes in the replicase gene [ ] , and that one or more of the ibv accessory gene products interfere with the hosts' interferon response [ ] [ ] [ ] . targeted rna recombination is another reverse genetics approach, so far only developed for mammalian coronaviruses from the genera alphacoronavirus and betacoronavirus [ , , ] . this system is based on the exchange of the spike gene by that of a coronavirus with a different host tropism, which enables subsequent selection on cells susceptible to the heterologous species [ ] . as a consequence, manipulation is limited to the last third of the coronavirus genome, covering all genes encoded that are located ′ of gene , starting with the spike gene. targeted rna recombination has been shown to be easy in use and to allow the rescue of highly defective mutants [ , , ] . however, the system is based on the ability to propagate both the donor and the recipient coronavirus in cell culture, and is hence not implementable for pathogenic ibv. this problem was solved by transfecting ibv genomic rna into otherwise non-susceptible cells, exchanging the ibv spike gene by that of the mouse hepatitis virus (mhv) provided as part of a synthetic rna, and by subsequently rescuing recombinant ibv from infected/ transfected cells in embryonated eggs (fig. ) . this system was successfully established to introduce marker mutations in the last one-third of the genome of ibv. the resulting recombinant viruses demonstrated growth kinetics in ovo and the in vivo phenotypic characteristics in one-day-old chickens similar to ibv wild type. the results presented here demonstrate for the first time a host species switch for an avian gammacoronavirus by exchanging the spike gene with that of the highly divergent betacoronavirus mhv. this rgs enables the manipulation of the structural and accessory protein genes from the genome of virulent ibv. baby hamster kidney (bhk- ) cells (atcc ccl- ) and murine lr cells kuo were cultured in dulbecco's modified eagle medium (dmem) (biowhittaker), supplemented with mm l-glutamine (lonza, basel, switzerland), % fetal bovine serum (fbs) (biowhittaker) and . mg/ml gentamicin (gibco invitrogen), at °c and % co . virus titers in cells were obtained by determining the % tissue culture infective dose (tcid ) per ml at days post inoculation (p.i.) according to the spearman-kärber method [ ] . fertilized specific pathogen free (spf) white leghorn eggs (animal health service, deventer, the netherlands) were incubated at . °c and - % relative humidity. a b c d fig. coronavirus genome organization and schematic overview of targeted rna recombination. a schematic genome representations of ibv (blue) and mhv (red). the first two-third of the genome is truncated, the structural and accessory genes are drawn to scale. the lengths of the first two-thirds and last one-third of the ibv genome are indicated at the top. the different domains of the spike gene are indicated: ss = signal sequence; ec = ectodomain; tm = transmembrane domain; en = endodomain. pcr amplicons are depicted as black bars drawn to scale above the genomes, with encircled letters referring to the primer sets in table and fig. . b stage in targeted rna recombination: an interspecies chimeric murinized ibv with a mhv spike ectodomain (mibv) is generated by a single recombination event of ibv genomic rna with synthetic rna transcribed from donor plasmid p-mibv in the ′-end region of the b gene (indicated by a black curved line). murinized ibv is selected on murine lr cells. plasmid inserts are indicated above p-mibv, with numbers in black circles referring to the plasmid junctions. c stage in targeted rna recombination: a recombinant ibv with the ibv spike gene (ribv) is recreated by a single recombination event of mibv with synthetic rna transcribed from donor plasmid p-ibv. recombinant ibv is selected on embryonated chicken eggs. d nucleotide sequences of the plasmid junctions, marked with corresponding numbers in the schematic donor plasmid drawings. nucleotide sequences are indicated for wild-type ibv and donor plasmids p-ibv and p-mibv, with restriction enzyme sites in italics, mhv spike gene sequences in lower case, and spike domains (i.e. ss, ec, tm and en) separated by vertical dashes. stop codons are highlighted in red. open reading frames (orfs) are underlined, overlapping orfs are double-underlined, and orf translations are indicated as amino acids below the nucleotide sequences if applicable eight-day-embryonated chicken eggs (ece) were inoculated via the allantoic cavity unless stated otherwise, and candled twice daily. upon embryonic death or no later than days p.i., eggs were transferred to °c for - h prior to allantoic fluid (af) and chorio-allantoic membrane (cam) collection. virus titration in ovo was based on the determination of the % embryonic infectious dose (eid ) per ml, as determined at day p.i. according to reed and muench [ ] . for the production of a virus stock, ten -day-old ece were inoculated with eid , incubated for h, and subsequently cooled for - h before the af was harvested and pooled. ibv strain h (boehringer ingelheim (bi), ingelheim, germany) was propagated in embryonated spf eggs and titrated. ibv strain beaudette (animal health service, deventer, the netherlands) was propagated and titrated in bhk- cells. mouse hepatitis virus (mhv) strain a was propagated and titrated in lr cells. monoclonal antibody (mab) ch/ibv . against the ibv s protein was obtained from prionics (thermo fisher scientific, waltham, ma, usa) [ , ] . the production of rabbit polyclonal antiserum k against mhv was described previously [ ] . chicken polyclonal antiserum was derived from a spf chicken vaccinated with ibv strain h (bi, ingelheim, germany). secondary fluorescently-labeled antibodies alexa fluor goat anti-chicken igy, alexa fluor goat antirabbit igg, and alexa fluor goat anti-mouse igg (invitrogen by thermo fisher scientific) were stored in % glycerol at − °c. cams were collected from eces, washed in pbs, fixed in neutral buffered % formalin in pbs for h, stored in % ethanol and finally paraffin-embedded. four micrometer sections of cam were mounted on glass slides and subsequently deparaffinized and rehydrated in alcohol series. next, the sections were subjected to endogenous peroxidase inactivation and antigen retrieval as described before [ ] . sections were washed in phosphate buffered normal antibody diluent (nad, scytek laboratories, logan, usa) containing . % tween- , and after primary antibody incubation with pbs . % tween- . sections were incubated for min at room temperature with mab ch/ibv . diluted : in nad. antibody binding was detected by dako envision hrpo labeled polymer anti-mouse (dako, by agilent technologies, santa clara, usa) diluted : in nad, and visualized by -amino- -ethylcarbazole (aec, dako). slides were counterstained with hematoxylin, mounted with aquatex (merck, darmstadt, germany), and viral antigen presence was assessed by light microscopy (bx , olympus, tokyo, japan). rna was isolated from harvested af using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to manufacturer's protocol. reverse transcription (rt) was performed using the transcriptor first strand cdna synthesis kit (roche, basel, switzerland) according to manufacturer's protocol, with random hexamers for standard pcr, or with specific primers for sequencing and cloning purposes. pcr was performed with recombinant taq dna polymerase (thermo fisher scientific) for plasmid characterization or with phusion hot start ii high-fidelity dna polymerase (thermo fisher scientific) for sequencing and cloning purposes. one-step rt-qpcr was used to semi-quantitatively assess virus load in af. forward primer ibv.rdrp.f ( ′-catgcagtttgttggagatcct- ′) and reverse primer ibv.rdrp.r ( ′-gtgacctggttttaccgt ttga- ′) targeting the conserved region of gene b (nucleotide position , to , in ibv beaudette genbank accession number m . ) coding for the rna-dependent rna polymerase protein. primers were obtained from biolegio (nijmegen, the netherlands) and used at a final concentration of nm each with the itaq universal sybr green one-step kit (bio-rad laboratories, hercules, california, usa). the rt-qpcr reaction was carried out in a bio-rad cfx connect realtime pcr system, starting with min at °c and min at °c, followed by cycles of s at °c and s at °c, and ending with a dissociation step for the determination of the melting point of the obtained pcr fragment. the complete genome sequence of ibv h bi was determined by sanger sequencing using primers as described by zhou et al. [ ] . the ′-and ′-utr sequences were identified using the nd generation ′/ ′ race kit (roche, basel, switzerland). the ibv h bi genome sequence was , nucleotides (nt) in length, including an annotated nt polya tail. the design of the donor plasmids principally followed the strategy previously described by kuo et al. [ ] . the final donor plasmid p-ibv was constructed from the stepwise ligation of fragments derived from five plasmids (fig. b and table , and described below in detail): plasmid (p)ibv- comprises a t rna polymerase promotor, g nucleotides, and the near full-length ′-untranslated region (utr), with an unintended a to c substitution at position . plasmid ibv- b comprises the last nt of the pol b gene, including the nt overlap with the spike gene, and the first nt of the spike gene, including the signal sequence. plasmid ibv-s contains the near full length ectodomain of the spike gene, nt in length. plasmid ibv-sir comprises the last nt of the spike gene (the transmembrane and the endodomain), the accessory gene , the envelope gene, the membrane gene and half of the intergenic region. plasmid ibv- t comprises the ′-terminal region of the ibv genome, including the second half of the ir, the accessory gene , the nucleocapsid gene, the ′-utr and a nt poly-a sequence. all plasmids were generated by gen-script (piscataway, nj, usa) and provided in the plasmid puc -simple, a standard cloning plasmid with the polylinker removed. in order to allow cloning of the fragments in a stepwise approach, naturally occurring restriction enzyme sites (res) located in the viral cdna were used, except for the bstbi site between p-ibv- and p-ibv- b, which is only partly present in the ′-utr, and the xhoi site between p-ibv- b and p-ibv-s, which was introduced without changing the amino acid sequence (silent mutation). restriction enzyme sites were made unique by silently removing these res from other parts of the genome included in the donor plasmid (additional file : table s ). in addition, semi-unique res were introduced by silent mutations within nt up-and downstream of the accessory genes and . finally, unique res mssi and paci were included after the poly-a sequence, allowing linearization of the plasmid by a single restriction enzyme digest. all genome fragments were ligated step-by-step into p-ibv- using the restriction enzymes specified in table . each ligation mixture was subsequently transfected into hb competent cells and plasmid dna was isolated by performing midiprep dna isolation (qiagen, hilden, germany). the final plasmid consisted of p-ibv- - b-s-sir- t, now called p-ibv (fig. c) . the composition of each plasmid was confirmed after each cloning step by pcr, restriction enzyme digestion and sequencing of each of the inserts (macrogen, amsterdam, the netherlands). the ectodomain of the mhv a spike gene was amplified from ptug [ ] by pcr using primers with an xhoi overhang (table ) and ligated into pjet . resulting in p-mhv-s. site directed mutagenesis (sdm) with the q sdm kit (new england biolabs, ipswich, usa) was used to silently remove an ecori and an xhoi res interfering with subsequent cloning steps ( table ). the ectodomain of mhv spike was ligated into p-ibv- - b, followed by subsequent cloning steps using the ibv fragments sir and t. this resulted in the plasmid p-ibv- - b-mhvs-sir- t, now called p-mibv (fig. b) . a plasmid comprising the nucleocapsid gene and ′-utr sequence of ibv h bi was generated by pcr amplifying the respective region using primers ibv-h .n.atg.fw and ibv-m # .ir.rv ( table ). the amplicon was ligated into pjet . downstream of the t promotor sequence, resulting in p-ibv-n, and the correctness of the insert was verified by sequencing. capped, run-off donor transcripts were synthesized from p-ibv, p-mibv and p-ibv-n using the mmessage mmachine t kit (ambion by thermo fisher scientific). in brief, p-mibv was paci-linearized, and p-ibv and p-ibv-n were mssi-linearized. linearized plasmid dna was ethanol precipitated. transcription reactions were prepared according to the manufacturer's instructions, using . and . μg linearized dna per ul reaction for p-(m)ibv and p-ibv-n, respectively. after h of incubation at °c, production of rna was verified by analyzing μl of the reaction volume by gel electrophoreses. after an incubation of h the reaction was stopped by transferring the reaction tubes to ice. targeted rna recombination and rescue of mibv the ibv spike gene was replaced by a chimeric mhv-ibv spike gene in the ibv genome by targeted rna recombination between p-mibv generated donor rna and recipient virus (ibv) rna, as described before [ ] . ibv h viral rna was transfected into bhk- cells, a cell line known to support replication of ibv [ ] , but not infection with ibv h (data not shown). thus, μl of ibv h bi rna obtained from allantoic fluid, mixed with μl transcript reaction mixtures of p-mibv and p-ibv-n each were transfected into bhk- cells by electroporation using two pulses at v and μf in a gene pulser electroporation apparatus (bio-rad). transfected bhk- cells were seeded onto monolayers of lr cells having an approximate confluence of - % and incubated at °c. two days after seeding, when syncytia in the lr monolayer were observed, the cell culture supernatant was harvested and rescued viruses were purified by two rounds of plaque purification on lr cells. characterization of the last one-third of the genome of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the obtained cdna-fragments, using the primer sets specified in table . murinized ibv (mibv) strain # b -iia was selected based on sequence analysis, and virus stocks were propagated and stored at − °c. aliquots were titrated on lr cells. recombinant ibv (ribv) was generated by substituting the ibv spike ectodomain back into the mibv genome by targeted rna recombination between p-ibv-generated donor rna and recipient virus mibv. lr cells were infected with mibv at a multiplicity of infection (moi) of . for h. capped, run-off donor transcripts from p-ibv were transfected into the mibv-infected lr cells by electroporation with two pulses at v and μf. electroporated lr cells were resuspended in ml dmem (at °c) and tenfold dilutions (up to − ) were prepared. two hundred microliters of lr cell suspensions were inoculated into the allantoic cavity of -day-old eces, using eggs per dilution. the eggs were candled twice daily and scored for embryonic death. upon death, or at days p.i., the eggs were transferred to °c. sixteen to twenty-four hrs later the af was collected aseptically for rt-qpcr, and the cams were fixed in % formalin for immunohistochemistry (ihc). the af from eggs inoculated with the highest dilution of electroporated lr cells, in which virus was detected by rt-qpcr and ihc, was subjected to two additional rounds of end-point dilution in -day-old ece. genetic characterization of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the region encoding the structural and accessory genes using the primer sets specified in table . biological characterization of the chimeric nature of mibv was performed by immunofluorescence (if) double staining for ibv and mhv. bhk- and lr cells were grown on coverslips and inoculated with ibv beaudette, and mhv a and mibv # b -iia, respectively, at an moi of . . cells were fixed with pbs % paraformaldehyde (aurion, wageningen, the netherlands) for min at room temperature after ¼, , and hpi for mhv, ibv, and mibv, respectively. subsequently, cells were permeabilized with pbs containing . % triton x- , blocked with goat serum (gibco by life technologies), and incubated for - min with a combination of two primary antibodies in ngs; rabbit anti-mhv polyserum k diluted : and chicken anti-ibv-h serum diluted : , or rabbit anti-mhv polyserum k diluted : and mouse mab ch/ibv . animals were housed in separate groups and inoculated via eye-drop with eid in . ml of ibv h bi (n = ), ribv-wt (n = ), or not inoculated (n = , negative control). clinical symptoms monitored included ruffled feathers, decreased consciousness, depression, gasping, coughing, tracheal rales, and nasal discharge. seven days p.i. animals were euthanized, and evaluated for their tracheal ciliary activity. to this end, the trachea was sliced into transversal sections: from the upper part, from the middle part, and from the lower part. ciliary activity was examined by low-magnification microscopy within h after sampling. ciliostasis of each tracheal section was scored on a scale from ( % ciliary activity) to (no ciliary activity, i.e. complete ciliostasis), with the maximum score for each trachea being . finally, the mean ciliostasis score for each group of animals was calculated. a unpaired t-test was performed to analyse whether there were differences in ciliostasis scores between ibv h bi and ribv-wt. generation and antigenic characterization of mibv and ribv-wt viral rna of ibv h bi and rnas transcribed from plasmids p-mibv and p-ibv-n were co-transfected into bhk- cells and seeded onto monolayers of lr cells. at days post transfection, syncytia were observed in the lr monolayers, suggesting the successful generation of recombinant mibv. after two rounds of plaque purification on lr cells, candidate recombinants were characterized antigenically and genetically. if staining of lr cells infected with mibv showed positive staining with both anti-ibv and anti-mhv sera, indicating the chimeric nature of mibv (fig. a) . if staining with an anti-ibv-s mab was positive for ibv beaudetteinfected bhk- cells (taken along as positive control for if), but not for lr cells infected with mibv, indicating the absence of ibv s protein in mibv (fig. b) . lr cells infected with mibv and subsequently transfected with rna transcribed from plasmid p-ibv were inoculated in tenfold dilution series into the allantoic cavity of -day-old eces. no embryonic death was observed up to days p.i., but embryos in the lowest dilutions showed signs of stunting and curling typical for embryos infected with ibv. the presence of replicating recombinant ibv was demonstrated by rt-qpcr on viral rna extracted from the af (data not shown), and by ihc on cam tissue (fig. c) . in contrast, cams of eggs inoculated with mibv-infected lr cells (not transfected) did not show any positive signal for ibv in ihc. during the first and second passage of ribv-wt in eight-day-old eces for endpoint dilution purposes, infected embryos died between and days p.i. using specifically located primers ( fig. and table ) , the intended genome structure and sequence of mibv and recombinant ibv wild-type (ribv-wt) were verified by rt-pcr (fig. ) . the most important findings were that ( ) murinized ibv contained the correct ′ s gene sequence (primer set [f] -with a forward primer located in the ibv b gene at a position upstream of the b sequence present in p-mibv and a reverse primer located in the mhv spike gene -resulted in a detectable pcr product for mibv, but not for ibv or p-mibv); ( ) murizined ibv contained the mhv spike gene at the location of the ibv spike gene (as both primer set [f] and [h] -each with a primer in the mhv spike gene and one in ibv gene b [f] or in the m gene [h] -resulted in a detectable pcr product for mibv, but not for ibv or mhv); ( ) the ibv spike gene was absent from the mibv genome (as primer set [d] targeting the ibv spike gene resulted in a detectable pcr product for ibv, but not for mibv or mhv( ); recombinant ibv was the result of recombination between genomic rna from mibv and rna transcribed from p-ibv (as primer set [c] -with a forward primer in ibv gene b located upstream of the b sequence present in p-mibv and a reverse primer located in the ibv spike gene -resulted in a detectable pcr product for ribv-wt, but not for mibv or p-ibv); ( ) recombinant ibv contained the ibv spike gene at the location of the mhv-derived spike in mibv (as both primer set [c] and primer set [e] -each with one primer in the ibv spike gene and the other in ibv orf b [c] or the m gene [e] -resulted in a detectable pcr product for ribv-wt, but not for mibv or mhv); ( ) the mhv spike gene was absent from the ribv-wt genome (as primer set [g] targeting the mhv spike gene resulted in a detectable pcr product for mhv and mibv, but not for ribv-wt). sequence analysis of the ′ kb of the mibv genome (starting kb upstream of the bstbi res in gene b, which marks the start of p-mibv) confirmed the expected genetic identity of mibv as observed after rt-pcr analysis (additional file : figure s ). the ′ kb of mibv and ribv-wt were exactly as designed, including the deliberate synonymous mutations listed in additional file : table s . a single spontaneous silent mutation (t to c) was observed in the spike of ribv-wt at position , . in ovo growth kinetics of ibv and ribv-wt were assessed after inoculating eces with eid per egg by determination of the relative viral load in the af of five eggs per virus at , , , and hpi by rt-qpcr. at hpi, the parental wild-type ibv showed somewhat higher viral loads as compared to ribv-wt, while from hpi onwards, viral loads were comparable for both viruses (fig. a) . the virus titers remained at the same level until embryos started to die between and hpi. no differences in embryonic death between ribv-wt and ibv h bi groups was observed ( fig. b ; p > . ). the pathogenicity of ribv-wt was compared to that of the parental ibv h bi strain by inoculating one-dayold spf chickens. during the course of the infection, no clinical symptoms were observed in any of the groups (data not shown). at days p.i. the animals were euthanized and the mean ciliostasis scores were determined as a readout for the ability of the respective viruses to infect and cause lesions in the primary target organ, the trachea. as expected, the negative control animals scored very low (fig. ) , while ibv h bi infection resulted in a score of . ribv-wt had a mean score of (p > . ), indicating that ribv maintained the ability to a b c fig. antigenic characterization of mibv and ribv-wt. a immunofluorescence analyses of ibv beaudette, mhv a and mibv # b -iia infected cells. lr cells infected with mibv were fixed and double-immunolabeled with a polyclonal against ibv (green) and a polyclonal antibody against mhv (red). ibv beaudette-infected bhk- cells and mhv-infected lr cells were taken along for comparison. nuclei are visualized with dapi (blue). overlay pictures (merge) are shown on the right. b similar to (a), except that a monoclonal antibody against ibv s was used instead of a polyclonal against ibv, indicating the absence of ibv s protein in mibv infected cells. c immunohistochemistry of ibv h bi and ribv-wt infected cam tissues. ten-day-old embryonated chicken eggs were inoculated with ibv h bi (positive control), mibv-infected and p-ibv transcript electroporated lr cells (resulting in generation of ribv-wt), mibv infected and p-ibv transcript, but not electroporated, lr cells (mibv + p-ibv mock) or pbs (mock). formalin-fixed and paraffin-embedded cam tissues were immunohistochemically stained using a monoclonal antibody against ibv s . replication of (r)ibv in the epithelial cells of the cam is indicated by red cytoplasmic staining, which is absent in eggs inoculated with mibvinfected non-transfected lr cells infect one-day-old chickens and induced lesions to a similar extent as the parental virus strain. here we developed a novel rgs based on targeted rna recombination, which allows manipulation of the genome of virulent ibv. the resulting recombinant virus has the same characteristics as the wild type ibv h both in embryonated eggs and in one-day-old chickens. by adapting the classical targeted rna recombination approach [ , , ] to ibv, the inability to culture ibv strains like h on continuous cell lines has been overcome. for this, a cell-line known to support the replication, but not the entry of, ibv was used. this observation was used to create, by co-transfection of ibv viral rna and a transcript of chimeric ibv carrying the mhv spike gene, the recombinant mibv virus. upon transfection of synthetic ribv donor rna into mibv-infected murine lr cells, subsequent infectious ibv virus particles could be rescued in ece. the feasibility of the approach was demonstrated by the generation of recombinant ribv virus carrying silent marker mutations. the inability to select for individual ibv recombinants by plaque purification was circumvented by a combination of three approaches. first, the mibv-infected and ribv donor rna-transfected lr cells were inoculated into eces by end-point dilution. second, early rt-pcr and sequencing based screening of the genetic make-up of recombinants helped to identify and discard erroneous recombinants. third, two subsequent end-point dilution series were executed in eces, each leading to the selection of ribv-wt. finally, the genetic identity of the ′ kb of ribv-wt, i.e. the part of the ibv genome had been subject to manipulation, was confirmed by sequence analysis. the replication and pathogenicity of ribv-wt in ece was comparable to that of the parental ibv h bi. viral loads in the af were similar with respect to maximum virus titers (fig. ) and embryonic death induced by both viruses did not differ (not shown). the pathogenicity of ribv-wt in one-day-old chickens was also comparable to that of parental ibv h bi, as demonstrated by comparable mean ciliostasis scores at days p.i. (fig. ) . taken together, ribv-wt has the same properties as ibv h both in ovo and in vivo and can thus be used to provide insights in the infection process in chickens. previously described rgs based on non-pathogenic ibv [ ] [ ] [ ] [ ] [ ] can be used for in vivo studies only upon introduction of virulence factors including spikes from other ibv serotypes [ ] [ ] [ ] . our newly developed rgs for ibv-h directly allows the elucidation of factors that determine the pathogenicity of ibv, as well as studying its protective immunity in vivo. fig. genetic characterization of wild type viruses, recombinant viruses and donor plasmids. pcr was performed on cdna templates of viral rna extracted from infected lr cell culture supernatants (mhv and mibv) and allantoic fluid of inoculated embryonated eggs (ibv and ribv-wt), plasmid dna (p-mibv and p-ibv), or no template control (−).primer set letters a to h correspond with letters depicted in fig. ; detailed information on the primers is given in table here, for the first time host species switching of an avian gammacoronavirus to mammalian cells was demonstrated, by exchanging the spike gene with that of the betacoronavirus mhv. manipulation of coronavirus genomes by targeted rna recombination using an interspecies chimeric coronavirus has been demonstrated for alpha-and betacoronaviruses, thereby switching species tropism between mammalian hosts. our observation confirms the spike gene as the principal determinant of host species tropism of both avian and mammalian coronaviruses. in summary, a novel reverse genetics system (rgs) based on targeted rna recombination that allowed manipulation of the genome of virulent ibv was developed. this system makes use of an interspecies chimeric coronavirus, which is created by replacing the ectodomain of the ibv spike protein by that of mhv. the spike ectodomain exchange results in a host species tropism switch, which enables replication of mibv in cell culture. upon recombination of mibv with synthetic donor rna carrying the ibv spike gene, ribv a b fig. in ovo characteristics of ibv and ribv-wt. a growth kinetics of ibv and ribv-wt were assessed by quantitative rt-qpcr analysis of rna extracted from allantoic fluid of inoculated embryonated eggs collected at , , , and hpi. data points represent means and standard deviations of eggs per condition, with all samples run and analyzed in triplicates, using a tenfold dilution series of ibv h bi as reference for quantification. b embryonic death is indicated as a percentage of all remaining animals at each time point could be rescued in eces. in ovo growth kinetics and in vivo characteristics were comparable for ribv-wt and parental ibv h bi, suggesting no attenuating effect of the recombination process or of the introduced synonymous marker mutations. this system will allow the introduction of mutations in the ′ one-third of the ibv genome, allowing the manipulation of the structural and accessory genes. the use of this system for both fundamental and applied research is promising, and potentially enables the development of a new generation of rationally designed live-attenuated ibv vaccines. additional file : table s . silent mutations introduced in ribv. nucleotide positions and sequences refer to the ibv h bi genome (see additional file : figure s ). modified nucleotides in recombinant ibv wt are depicted in lower case; n.a. = not applicable. purpose of introduction of restriction enzyme site are indicated for each site; in case of enzyme site removal the purpose was to create unique restriction enzyme sites for cloning; n.a. = not applicable in this study. (docx kb) additional file : figure s . alignment of ′ kb of mibv and ribv-wt with ibv h bi. alignment of the ′ kb of mibv b iia p (excluding the mhv derived spike ectodomain sequence) and recombinant (r)ibv wild-type (wt) p with ibv h bi. numbers refer to nucleotide positions in the ibv h bi genome. restriction enzyme sites are highlighted in yellow, with the corresponding enzyme indicated above the sequences. an additional thymidine residue to keep the mhv spike gene ectodomain sequence in frame with the ibv spike gene signal sequence at position , is highlighted in green and marked with a # above the sequence. the long view: years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures family coronaviridae in: ninth report of the international committee on taxonomy of viruses coronavirus avian infectious bronchitis virus coronaviridae accessory proteins of sars-cov and other coronaviruses gene of the 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alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus geert de vrieze, maartje woelders, maloeke de jong, and alexandra negatsch are acknowledged for excellent technical support. this research was financially supported by boehringer ingelheim, ingelheim, germany. data generated or analyzed during this study and described in this manuscript are included in this manuscript and its supplementary information files. authors' contributions sjvb, em, pjmr and mhv designed the studies. sjvb, ajb, akk, gc, ds, and hcp carried out the experiments. sjvb wrote the manuscript. mhv, em, and pr edited the manuscript. all authors read and approved the final manuscript. submit your next manuscript to biomed central and we will help you at every step: key: cord- - v gzhf authors: aponte, fernando e; taboada, blanca; espinoza, marco a; arias-ortiz, maría a; monge-martínez, jesús; rodríguez-vázquez, rubén; díaz-hernández, fidel; zárate-vidal, fernando; wong-chew, rosa maría; firo-reyes, verónica; del río-almendárez, carlos n; gaitán-meza, jesús; villaseñor-sierra, alberto; martínez-aguilar, gerardo; garcía-borjas, maricela; noyola, daniel e; pérez-gónzalez, luis f; lópez, susana; santos-preciado, josé i; arias, carlos f title: rhinovirus is an important pathogen in upper and lower respiratory tract infections in mexican children date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: v gzhf background: most of the studies characterizing the incidence of rhinovirus (rv) have been carried out in hospitalized children and in developed countries. in those studies, rv-c has been associated with more severe respiratory tract infections than rv species a and b. in this study we determined the frequency and diversity of rv strains associated with upper and lower respiratory tract infections (urti, lrti) in mexico, and describe the clinical characteristics of the illness associated with different rv species. methods: a prospective surveillance of and children with urti and lrti was carried out. respiratory samples were analyzed by rt-pcr for viruses. the ′ untranslated region of the rv genome was amplified and sequenced. results: in the case of urti, . % were positive for rv, while this virus was found in . % of lrti. the rv species was determined in children with urti: . % were rv-a, % rv-c and, . % rv-b; and in children with lrti: . % were rv-a, . % rv-c, and % rv-b. no significant differences in clinical characteristics were found in patients with rv-a or rv-c infections. a high genetic diversity of rv strains was found in both urti and lrti. conclusions: both rv-a and rv-c species were frequently found in hospitalized as well as in outpatient children. this study underlines the high prevalence and genetic diversity of rv strains in mexico and the potential severity of disease associated with rv-a and rv-c infections. pneumonia continues to be a major killer of young children in developing countries and elderly people in developed countries. despite recent advances, further studies are needed to examine regional variation in its etiology, particularly in developing countries, where most of the deaths from serious respiratory diseases occur [ , ] . rhinovirus (rv) is the most frequent cause of acute respiratory illness worldwide [ ] [ ] [ ] . this virus has been typically associated with upper respiratory tract infections (urti); however, with the development of molecular methods rv has been found to be also commonly associated with lower respiratory tract infections (lrti). these findings are changing the long held view of rv as a minor pathogen, as it is now being involved in a wide variety of respiratory illnesses, ranging from mild common colds and asthma exacerbation to serious lower respiratory tract disease [ ] [ ] [ ] . rvs are antigenically quite diverse; historically, these viruses were classified into serotypes through neutralization assays. these different rv serotypes were later classified genotypically into two species, rv-a ( serotypes/genotypes) and rv-b ( serotypes/genotypes) [ ] . more recently, based only on genomic sequence information, a third virus species, rv-c, was described [ ] [ ] [ ] [ ] . the current genotypification of these viruses is based on the nucleotide sequence encoding the vp and vp /vp proteins [ ] . nevertheless, a variable region in the ′-untranslated region (utr) of the viral genome can also accurately distinguish among virus species [ ] . since its description in , rv-c was suggested to be associated with more severe respiratory illness as compared to rv-a and rv-b, as well as with more frequent asthma exacerbations [ ] [ ] [ ] [ ] [ ] [ ] . however, other studies have found no difference in illness severity among rv species [ ] [ ] [ ] [ ] , thus, more information is needed to clarify this issue. most studies have described the presence of rv genotypes in hospitalized patients with severe respiratory illness, and only a few studies have described the prevalence of virus genotypes in urtis. in this work, we carried out a prospective multicenter study of two children populations having either urti or lrti. nasopharyngeal samples from pediatric patients with urti attending the private consult were tested for the presence of rv and other respiratory viruses. ninety-two ( . %) children ( males and females, age range to months) were rv positive. the species of ( . %) of these rvs was determined by sequencing the ′-utr region of the viral genome; ( . %) were rv-a, ( %) rv-c, and ( . %) was rv-b (table ). the incidence of rv infection was highest in september and november as well as in april , when it accounted for . %, . % and . %, respectively, of the total samples collected ( figure ). on the other hand, hospitalized children with clinical diagnosis suggestive of viral pneumonia were included in the study. nasal washings were obtained and tested for the presence of respiratory viruses. sixty-two children ( males and females, age range to months) were positive for rv ( . %). the species of ( . %) of these viruses was determined by sequencing the '-utr region of the viral genome. of the typed viruses, ( . %) were rv-a, ( . %) rv-c, and ( %) were rv-b (table ). the difference in the incidence of rv-a and rv-c was not statistically significant. rv-positive children were detected more frequently (p < . ) in children between and months as compared to the other age groups (table ). there was no significant gender between children infected with rv or another respiratory virus or between rv-a and rv-c. hospitalization for rv was significantly higher (p < . ) in the spring as compared to other seasons. an additional respiratory virus was found in % ( / ) and % ( / ) of rv-positive patients with urti and lrti, respectively ( table ) . the virus species could be determined in ( rv-a, rv-c) and ( rv-a, rv-b, rv-c) of urti and lrti mixed infections, respectively. neither rv-a nor rv-c types were significantly associated with a particular respiratory virus in mixed infections of urti or lrti. however, in urti, rv-a was found more frequently in co-infections than rv-c (p = . ). in both children populations, when rv was present in mixed infections, it was more frequently found associated with a single virus (urti, / ; lrti, / ) than with other viruses (urti, / ; lrti, / ) (urti p = . , lrti p < . ). the major symptoms and signs observed in children with rv-associated urti in single or mixed infections were cough, rhinorrhea, and sore throat. however, rhinorrhea was the only symptom significantly higher in rv-positive children as compared to patients with infections caused by other respiratory viruses ( table ). increased respiratory rate was found in more than one third of the patients with a single or mixed infection with rv, as well as in infections caused by other viruses. the clinical features of children having a co-infection with rv and other respiratory virus were no statistically different from those with single rv infections. also, no significant difference was found between patients infected with rv species a or c. rv-b was excluded from the analysis given that a single child was infected with this virus. in . % of the rv-positive patients with lrti the diagnosis of pneumonia was confirmed radiologically, as it was in . % of children positive for other viruses (table ). children with rv had fever > °c and increased respiratory rate more frequently than children with any other respiratory virus. this was true for single and mixed rv infections. other major signs detected upon examination of rv-positive patients were thoracoabdominal dissociation, and intercostal retraction. according to the silverman-anderson score there was no difference between children with rv or any other virus; . % of the patients with rv co-infections and . % of rv single infections had a score between and . in the chest-x ray examination, . % of the rv-positive patients had signs of interstitial pneumonia; these percentages were similar for rv-single infections. there was no difference between the clinical signs observed in children infected with either rv-a or rv-c. the phylogenetic trees in figure depict the wide distribution of ′-utr sequences of the rv-a and rv-c table frequency of rv infections in children with upper and lower respiratory tract infections strains isolated from urti and lrti patients. the characterized rv strains could be classified into different ′-utr-based genotypes ( ′-genotypes); in this work, two ′-genotypes were defined as different when their ′-utr sequence identity was equal or less than %, similar to the cutoff determined for genotypes based on vp nucleotide sequences [ ] . seventeen genotypes were associated with rv-a and with rv-c. each genotype included from to different virus strains. in the case of rv-a, ′-genotypes were significantly more frequently found in either hospitalized (genotype , p < ) or outpatient (genotype , p < . ) children, while for rv-c some ′-genotypes also associated more frequently with either urtis (genotypes , p < . and , p = . ) or lrtis (genotype , p < . ) ( figure ) . importantly, the correlation of ′-genotypes with reference genotypes defined by full genomic sequences [ ] was remarkably high. comparison of the phylogenetic trees constructed with either the ′-genotypes or the full genomic sequences showed that % ( / ) of the terminal clades present in the ′-phylogenetic tree were the same as compared with the terminal clades of the reference tree ( figure ). these results suggest that ′-genotypes could serve as a simpler and initial approach to genomically classify rvs. further studies need to be conducted to determine if the association between some rv ′-genotypes and the severity of infection observed in this work holds, or if it is the result of particular rv genomic types that circulated more frequently in the years that urti and lrti samples were collected. here, we report the frequency of rv in pediatric patients with urti and lrti in different regions of mexico. the frequencies observed in this work are in agreement with those reported in other studies, in which rv has varied from % to % in children with either urti or lrti. regarding seasonality, rv infections were associated most frequently to urti in autumn and early spring, as reported [ ] , although it should de noted that the samples from patients with urti miss the months from may to august. on the other hand, a clear peak of prevalence of the virus in ltri was observed only during spring. an additional respiratory virus was found in about one-third of the rv-positive patients with either urti or lrti. detection of several viruses in a high proportion of cases has been a characteristic of respiratory infections in which a pcr-based diagnostic method was used. in particular, rv has been found in dual infections with another respiratory virus in % to % of cases [ , , ] . however, the clinical relevance of detection of several viruses in pneumonia, and the association with severe illness is uncertain [ , [ ] [ ] [ ] . despite technological advances, establishing the cause of pneumonia remains challenging [ ] . in this study diagnosis of viral pneumonia relied on nasopharyngeal specimens, what might be misleading since detection of a virus in the nasopharynx could represent a coincidental upper-respiratory infection, the asymptomatic presence of the virus, or a genuine pneumonia pathogen. measurement of background prevalence of asymptomatic nasopharyngeal viral infections in a healthy control group and in patients with mild-to-moderate disease might help to clarify the relevance of this diagnostic issue at a population level [ ] . in most of this type of studies, but not in all, a higher rv detection among participants with illness was noted [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . the overall identification of rv was similar but statistically different between the two patient populations studied. children with urti had a rv rate of % as compared to . % in patients with lrti (p < . ). the observation that the frequency of rv detection in serious respiratory disease is similar to that observed in mild-to-moderate illness, or even in asymptomatic cases [ , , , [ ] [ ] [ ] [ ] [ ] [ ] , is intriguing. one would think that if the respiratory disease caused by rv was more severe than other pathogens, the percentage of hospitalized children positive for rv should be significantly higher than that found in outpatient or healthy children, as observed, for instance, in rotavirus infections [ ] . on the contrary, the incidence of rv should have to be significantly lower in lrti as compared to urti if the virus infection caused a mild disease. then, why is there about the same incidence of rv found in respiratory infections with different degrees of severity? it is tempting to hypothesize that specific virus serotypes/genotypes are preferentially associated to severe or mild clinical symptoms. this potential association might be obscured by a wide diversity of circulating rv serotypes/genotypes with potentially different intrinsic virulence, such that in different children populations similar frequencies of rv infection are observed but with different clinical outcomes. it cannot be discarded, however, that mixed infections with bacteria (not searched in this work) or different host factors could influence the clinical severity of rv infections, as reported [ ] . of interest, there have been suggestions since the earliest epidemiological studies of variation of virulence among the different rv serotypes [ , ] and, in this line, we noted in this work that some ′-genotypes were detected only in either hospitalized or outpatient children. it would be important to characterize the diversity of rv genotypes in different parts of the world to determine if the prevalent viruses vary among regions and in time, and associate with different severity of the disease. identification of this potential association would have a great impact in the development of prophylactic measures to control the infection of this very common pathogen. in conclusion, this study underlines the high rv exposition and diversity of circulating strains in mexico and the potential severity of disease associated with both rv-a and rv-c infections. this study describes the frequency of detection of rhinovirus species in children with upper and lower respiratory tract infections in mexico and their genetic diversity, determined by sequencing the ′ utr region of the viral genome. we report that the ′utr sequence can be useful for an initial approach to determine the rhinovirus genotypes. we also describe the clinical characteristics of illness associated with specific rv species. we found that both rv-a and rv-c species were very frequently found in both hospitalized and outpatient children and no statistically significant differences were found in the severity of disease associated with rv-a and rv-c infections in neither of the two children populations. this study underlines the high rv prevalence and genetic diversity of circulating strains in mexico and the potential severity of disease associated with both rv-a and rv-c infections. this is of particular relevance, since the information about respiratory viruses in mexico is very limited, and studies characterizing viruses circulating in the community level are even scarcer. two patient populations were included in this study. the first was composed of pediatric patients that attended the private consult in five different cities of the state of veracruz, mexico (poza rica, veracruz, córdoba, tierra blanca, and minatitlán). the children were enrolled in the study if they were younger than years, clinically diagnosed as having an acute respiratory infection, with an onset of illness less than one week, and the parent or guardian signed the informed consent form. since none of these children required hospitalization, they were all considered as having urtis. from september to april nasopharyngeal swabs (rayon-tipped, bd bbl) were collected from children (male:female ratio, and stored at − °c until analyzed. the study was approved by the institutional review boards of the school of medicine and the institute of biotechnology of the national university of mexico and from the institutional review board and ethics committee of each participant hospital. written informed consent was obtained from each parent or guardian prior to enrollment. nucleic acids were extracted from μl of respiratory specimens using the purelink viral rna/dna mini kit (invitrogen, carlsbad, ca). rv and other fourteen respiratory viruses were detected with the seeplex®rv onestep ace detection kit (seegene, seoul, south korea) following the manufacturer's instructions. this diagnostic test detects the following viruses: parainfluenza viruses , , , and , adenovirus a/b/c/d/e, human coronaviruses e/nl and hucov-oc , rinovirus a/b/c, influenza a, influenza b, respiratory syncyctial virus-a and -b, human bocavirus / / / , human metapneumovirus, and enterovirus. the rna present in the samples positive for rv was reverse transcribed with random hexamers using standard protocols. pcr was then performed with previously described primers dk and dk , which target a fragment of approximately bp of the hypervariable region of the rv ′-utr virus genome [ ] . the amplified dna fragment was purified with the high pure pcr product purification kit (roche). the purified pcr products were sequenced in an applied biosystems (foster city, ca), model xl apparatus. the sequences obtained were edited using the unipro ugene and seaview softwares. a database of rv complete genomes was created using all sequences available on genbank until september . blast was performed and the genotype of the rv strains was assigned based on the first five best scores. the ′ sequences of the rv strains characterized in this work were deposited in genbank with accession numbers kj to kj . multiple sequence alignments were made using muscle method; maximum likelihood trees were generated with repetitions bootstrap using the mega . . program. to determine the ′-genotypes, clustering was made using the cd-hit-test at % of identity. all statistical data analyses were carried out using pasw statistical software version . (spss inc.). significant differences between groups were evaluated using chi-square tests when it was possible, or fisher's exact tests when values were smaller than . associations of demographic and clinical features of children with a) rv detected versus other respiratory virus and, b) rv-a versus rv-c, were examined using logistic regression. to determine the ′genotypes that were significantly enriched for rv-a or rv-c, the probability of randomly selecting different viral strains in each ′-genotype was calculated by a combinatorial approach. in all statistical analysis p-values < . were considered statistically significant. written informed consent was obtained from each parent or guardian prior to enrollment in the study. global, regional, and national causes of child mortality in : a systematic analysis viral pneumonia fields virology the common cold rhinoviruses: important respiratory pathogens sequencing and analyses of all known human rhinovirus genomes reveal structure and 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take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank arely garcía botello for her valuable help in integrating the databases used in this work. we also thank miguel l. garcía-león for his help in handling and organizing all pneumonia samples. this work was supported by grants (to j.i. santos) and "influenza " (to c.f. arias) from the national council for science and technology-mexico (conacyt). f.e.a. is recipient of a scholarship from conacyt. the authors declare that they have no competing interests. key: cord- -a cuq o authors: liu, jiangning; yang, yajun; xu, yanfeng; ma, chunmei; qin, chuan; zhang, lianfeng title: lycorine reduces mortality of human enterovirus -infected mice by inhibiting virus replication date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: a cuq o human enterovirus (ev ) infection causes hand, foot and mouth disease in children under years old and this infection occasionally induces severe neurological complications. no vaccines or drugs are clinical available to control ev epidemics. in present study, we show that treatment with lycorine reduced the viral cytopathic effect (cpe) on rhabdomyosarcoma (rd) cells by inhibiting virus replication. analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. lycorine treatment of mice challenged with a lethal dose of ev resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. when mice were infected with a moderate dose of ev , lycorine treatment was able to protect them from paralysis. lycorine may be a potential drug candidate for the clinical treatment of ev -infected patients. human enterovirus (ev ) is a positive-stranded rna virus belonging to the enterovirus genus of the picornaviridae family [ ] . ev infection predominantly leads to hand, foot and mouth disease (hfmd) in children under years old [ , ] . most of the ev infections resolve spontaneously; however, ev infection occasionally causes neurological complications that can lead to cardiopulmonary failure and death [ ] [ ] [ ] [ ] . outbreaks of ev have erupted around the world in the past decades and mainly appearing in the countries and regions of asia in recent years [ ] [ ] [ ] . hundreds of cases involving lethal complications have been reported in each outbreak [ , , ] , and the epidemic is still prevalent in asia [ ] . currently, there are no vaccines or antiviral drugs available to use against ev infection in the clinic, and the prevention of ev epidemics depends upon public surveillance alone. in recent years, there have been many efforts to develop drugs to combat ev infection and dozens of drugs have been reported to show anti-ev activity in vitro, some of which have been evaluated in animal models including bovine lactoferrin [ ] , ribavirin [ ] , sirna [ ] and type i interferon [ ] . although these drugs showed activity against ev infection both in cell lines and in animal models, the clinical application is not yet available. lycorine is one of the most abundant alkaloids of amaryllidaceae [ ] , and has a wide range of biological effects including apoptotic effects on tumor cells [ ] , antimalarial effects [ ] , anti-inflammatory effects [ ] and induction of nausea and emesis [ ] . importantly, lycorine also has been shown to have antiviral effects on human immunodeficiency virus (hiv- ), severe acute respiratory syndrome-associated coronavirus (sars-cov), poliovirus, coxsackie virus, measles virus, and herpes simplex virus type [ ] [ ] [ ] . in the present study, the activity of lycorine against ev replication was investigated in vitro and in mice models infected with ev strain. human rhabdomyosarcoma cells (rd) were maintained in dulbecco's modified eagle's medium (dmem) containing % fetal bovine serum (fbs) as previously described [ ] . a clinically isolated ev strain fy (genbank accession no. hq ) and the mouse-adapted ev strain mp (genbank accession no. hq ) derived from fy were cultured in rd cells. the virus titres were determined using a plaque assay as described [ ] , and working stocks of virus containing tcid /ml were prepared for experiments. for the antiviral assay, rd cells ( × cells/well) were plated in -well plates with dmem medium lacking antibiotics and grown overnight to % confluence at °c. the rd cells were then infected with tcid of fy and cultured continually in dmem medium containing % fbs. the infected cells were treated with lycorine (purity ≥ % in an hplc assay, national institutes for food and drug control) in a set of concentrations in saline as . , . , , , , and μg/ml. the infected rd cells were observed for cpe or harvested at eight-hour intervals post infection to determine the number of viral rna copies by quantitative rt-pcr (qrt-pcr). the half maximal inhibitory concentration (ic ) was defined as the concentration of lycorine that caused a % cpe reduction compared to that of the saline-treated control [ ] . the concentration of lycorine that was required for % cell cytotoxicity (cc ) was determined in rd cells as described previously [ ] . briefly, rd cells were plated in -well plates and grown overnight to % confluence at °c. the cells were then treated with lycorine for hours at °c in a set of concentrations between and μg/ml. cc was defined as the concentration of lycorine that caused % cpe of rd cells. to assess the expression of viral proteins following lycorine treatment, rd cells ( × cells/well) were plated in -well plates and grown overnight to % confluence at °c. the cells were then infected with tcid of fy and cultured continually for hours. the infected cells were treated with μg/ml lycorine. the infected rd cells were harvested after washing three times with pbs (ph . , gibco) at . , . and . hours of treatment. the harvested cells were resuspended in μl of pbs and lysed with three freeze-thaw cycles and the cell debris was then removed by centrifugation at , g for minutes. the supernatants were subjected to viral rna and protein analysis, and the infected rd cells treated with saline were used as control. for western blotting assay, equal amounts of aliquots ( μl of supernatants) were separated by % sds-page and transferred onto a nitrocellulose membrane (immobilon nc, millipore france). the polyclonal mouse antibodies against the viral peptides were prepared in our lab (anti-p - for vp , anti-p - for vp , anti-p - for vp , anti-p - for c, anti-p - for c, and anti-p - for d, : dilution) [ ] . primary antibodies were visualised with hrp-conjugated goat anti-mouse secondary antibodies (sigma) using a chemiluminescent detection system (santa cruz). the bands were quantified by densitometry using quantity one software. the intensity of bands for six viral proteins in the saline-treated group were used as standards and defined as . the density of the bands of viral proteins in the lycorine-treated group was compared to their respective standards. qrt-pcr was used to detect the viral rna copy number. briefly, total rna was isolated from cultured cells or tissues from mice using the trizol reagent. the total rna was then reverse transcribed using random hexamers with a reverse-transcription kit (promega). the cdna was subjected to quantitative pcr (quantitect sybr green rt-pcr kit, qiagen) with a roche light cycler . system for cycles. the primers were ev -s ( '-agatagggtggcagatgtaattgaaag- ') and ev -a ( '-tagcatttgatgatgctccaat ttcag- '). a fragment corresponding to nucleotides - of fy was adjusted to a concentration gradient ( × copies/μl to × copies/μl) and was used as standard to calculate the copy number of viral rna. results were normalised to gapdh. semi-quantitative rt-pcr was used to confirm the results of qrt-pcr. briefly, the viral cdna obtained above was assayed by pcr amplification with the same primers, and the results were normalised to gapdh. ten-or eleven-day-old icr mice were provided by the institute of laboratory animal science, peking union medical college. all of the animal protocols were approved by the institutional animal care and use committee. for lethal ev challenge, each ten-day-old mouse was intraperitoneally (i.p.) inoculated with × tcid (lethal dose) of mp . for self-limited model development, each mouse at eleven-day-old was inoculated with × tcid of mp via the i.p. route. at hours post infection, the infected mice were intraperitoneally injected with different concentrations of lycorine in saline daily twice for days. the placebo group was injected with the same volume of saline as control. the symptoms and survival rates of infected mice were monitored daily for weeks, the clinical scores were graded following previously described standards [ ] and the muscle tissues of mice were sampled at dpi, dpi, dpi, dpi and dpi and sent for virology and pathology analysis. for each experimental group, six mice were subjected to pathologic examination. after euthanasia, the muscle tissues were immediately immersion-fixed in % buffered formalin for hours. the tissues were bisected, embedded in paraffin, and stained with hematoxylin and eosin stain (h&e). ten sections of muscle were observed per animal in a blinded manner. immunohistochemistry immunohistochemistry (ihc) was used to detect virions in the infected tissues as described previously [ ] . briefly, the sections were incubated with a monoclonal mouse antibody for vp of ev (millipore, : dilution) for hour at °c. the sections were then washed three times with pbs and incubated with hrp-conjugated goat anti-mouse igg ( : dilution, sigma) for hour at °c. the sections were developed with - 'diaminobenzidine (dab) and observed under a light microscope (olympus). all data are expressed as the mean ± sd. the statistical significance of differences in mean values was assessed by duncan's multiple-range test following a one-way analysis of variance (anova), and survival rates were analysed by kaplan-meier analysis. a p value of < . was considered to be significant. the anti-ev efficacy of lycorine was tested on a human muscular (rd) cell line in a plaque reduction assay. as a positive control, the ic of ribavirin was determined in present study and was μg/ml [ ] . similarly, lycorine inhibited ev infection of rd cells in a dose-dependent manner with an ic of . μg/ml ( figure a ). and the replication of ev ( figure b) and majorities of the cpe in rd cells ( figure c to study the inhibitory mechanism of lycorine against ev infection, the synthesis of several typical viral proteins were detected by western blotting in . hours periods following lycorine treatment, when the effects of inhibition was just appeared ( figure a and b) . vp , vp , vp , c, c and d were sequentially translated from n-terminal to c-terminal of the viral polyprotein during viral proteins synthesis. the band densities of these proteins at . hour of saline-treatment were set as . then the densities of these proteins at . hour of lycorine-treatment were compared with that of saline-treatment respectively ( figure b and c) . the inhibition of lycorine on synthesis of c terminal proteins was more remarkable than that of n terminal proteins, as the inhibitory rate of vp was . %, whereas that of d was . %. this result suggested that lycorine affected the elongation of the viral polyprotein during translation. we utilized the mouse model of lethal ev infection to evaluate the effect of lycorine on inhibiting ev infection. the placebo-treated mice developed paralysis at dpi and all of them died within dpi, and treatment with ribavirin ( mg/kg body weight) enhanced the survival rate of infected-mice to %. meanwhile, the lycorine-treatment at doses of . mg/kg prolonged the survival time of mice; while the treatment at doses of . or . mg/kg enhanced the survival rates of mice to % respectively ( figure a ). this result revealed that . mg/kg was a proper dosage and this dosage was used to analysis the effect of lycorine against ev in the further experiments. the clinical scores of infected mice treated with placebo or lycorine ( . mg/kg) were systematically evaluated. treatment with lycorine delayed the paralysis appearance to day later compared with that of the placebo. and the surviving mice ( %) in the lycorine group were completely recovered within dpi ( figure b and c), while all of the mice in the placebo group were died within dpi. consistent with the results of the clinical scores and survival rates, virus replication in the muscle of lycorine-treated mice were inhibited by - folds at different time points compared to that of the saline control as detected by qrt-pcr and semi-quantitative rt-pcr ( figure a and b). lycorine treatment also obviously reduced the amount of virions in muscle tissues compared to the saline control by immunohistological staining ( figure a ). in the saline-treated group, serious muscle necrotic appeared at dpi and lead to paralysis of mice, while the lycorine-treatment ( . mg/ kg) obviously reduced the muscle damage caused by ev infection (figure b , moderate inflammation were observed in the muscle tissues of lycorine treated mice at dpi and dpi, contrast to the necrotising myositis of placebo-treated mice from dpi to dpi). lycorine treatment protected mice from apparent symptoms post non-lethal dose ev challenge the mouse model with lethal-dose ev infection was used to imitate the severe complications of ev infected patients. however, as most of the ev infection in patients leads to symptoms resolves spontaneously [ ] , we also explored the effect of lycorine against a moderate dose of mp infection in mice, which was supposed to imitate the self limited patients. the virus replication in muscle tissues of lycorine-treated ( . mg/kg) mice was significantly inhibited by more than -fold compared to the saline control as detected by qrt-pcr and semiquantitative rt-pcr ( figure a and b) . the salinetreated mice developed transient ruffled hair and paralysis and recovered within days. the lycorine treatment protected the infected-mice from symptoms of paralysis except occasionally skin fur ( figure c and d) . these results indicated that lycorine treatment was able to completely protect the mice from obviously symptoms upon a low-dose ev challenge. along with its broad biological activities and capacity to inhibit the replication of many different viruses, we confirmed that lycorine can inhibit ev infection. lycorine showed inhibitory activity against ev replication in rd cells, and lycorine treatment significantly enhanced figure lycorine treatment reduced the mortality of ev infected mice. a, survival rates of the ev infected mice treated with placebo, ribavirin and lycorine (three different doses) were recorded to dpi (n = ). *: p < . , **: p < . (survival rate of the mouse after . mg/kg lycorine treatment compared to that of the placebo). b, the clinical scores of infected-mice treated with placebo or lycorine ( . mg/kg) was systematically evaluated in an independent experiment (n = ). *, p < . . c, photos were a typical phenotype of ruffled hair and paralysis of hind limbs caused by ev infection at dpi (left panel, indicated by arrow) and the phenotype was prevented by the lycorine treatment (right panel). the survival rate of ev -infected mice, which demonstrates that lycorine may be a potential drug candidate for the clinical treatment of ev infection-induced diseases. previous studies indicated that lycorine can inhibit protein synthesis in eukaryotic cells [ , ] and in cellfree systems in which protein synthesis is catalysed by eukaryotic ribosomes [ ] . however, there have debates on which step of protein synthesis is blocked by lycorine. jimenez et al. has indicated that lycorine halts protein synthesis in eukaryotic cells by blocking the peptide bond formation [ ] . lycorine was shown to prevent the coupling of the n-acetyl leucyl residue from uaccaacetyl leucine to puromycin, and it was therefore postulated that these compounds inhibited the transpeptidation reaction [ ] . in contrast, it has been reported that lycorine affects the termination but not the elongation nor the cleavage of the polyprotein [ ] . the single large coding region of the ev genome is flanked by ' and ' untranslated regions ( ' and ' utr). the coding region is translated as a single polyprotein, which is then processed by viral proteases to yield mature viral proteins including the capsid proteins (vp -vp ) and non-structural proteins ( a- c and a- d) [ ] . we investigated the inhibitory mechanism of lycorine on ev replication by detecting the step of the viral life cycle that was initially blocked post drug treatment and found that the repression effect of lycorine on the synthesis of viral proteins located at c terminal of the polyprotein was earlier and more substantial than its effect on viral proteins located at n terminal of polyprotein ( figure ) ; therefore, we concluded that the drug inhibits the elongation of the viral polyprotein during protein synthesis. the imbalanced synthesis of viral proteins could interrupt the package of the virus. and the inhibition of d protein could result in the reduction of replication of virus, as the d protein is the rna polymerase of ev [ ] . in contrast to its other wide range of biological effects, lycorine is suspected to be the source of amaryllidaceae poisoning in humans and animals [ ] . empirical data from previous reports suggests that lycorine may be responsible for symptoms such as nausea and emesis [ ] . high concentration of lycorine-induced apoptosis of rd cells was observed in this study (figure ), which indicated that lycorine is noxious to eukaryotic cells. however, lycorine could inhibit the replication of ev at very low concentration and the ic for inhibiting ev infection was approximately -fold lower than the cc on rd cell, suggesting that it had potential application in the clinical treatment of ev infection. this assumption was verified by evaluating the antiviral effect of lycorine in a mouse model, as the survival rate of . mg/kg lycorine-treated mice was significantly enhanced compared to the control mice upon lethal ev infection ( figure a) , and the alleviation of acute symptoms and reduction of pathological changes in the muscles of lycorine-treated mice were achieved by inhibiting the replication of virus (figure , , ). the surviving mice in the lycorine treated group recovered within days. furthermore, lycorine treatment did not cause figure lycorine inhibited the replication of ev in muscle tissues of mice. the infected-mice were treated with placebo or lycorine at a dose of . mg/kg. the muscle tissues were sampled at dpi, dpi, dpi and dpi respectively (n = ). a, the viral replication curve were determine by qrt-pcr (*: p < . ). b, the viral burden was confirmed by semi-quantitative rt-pcr. any obvious side effects in the mice at the tested doses (data not shown). following lethal ev infection, the lycorine-treated mice had a survival rate that reached %, while all of the saline-treated mice were died within dpi ( figure ) . in previous studies, treatment with several drug candidates, including ribavirin, bovine lactoferrin, sirna and type i interferon, were able to enhance the survival rates of infected mice to %- % [ ] [ ] [ ] [ ] . however, because the infection doses of virus and strains were different in these experiments, it is difficult to compare the activity of these drugs, but we observed that the efficiency of lycorine against ev infection was better than ribavirin, as shown in ic determination ( figure a ) and evaluation by a mouse model ( figure a ). furthermore, as most of the ev infections were selflimited and did not cause severe complications in patients, self-resolved ev infection model, in which the infected mice develop paralysis at dpi and the symptoms were basically self-limited within dpi. as we expected, lycorine treatment was able to protect the infected-mice from paralysis symptoms by inhibiting ev replication (figure ) . meanwhile, we found that lycorine could also inhibit the infectivity of three other ev strains (these strains were isolated from clinical specimens from different regions of mainland china and belong to the c genotype) on rd cells (data not shown); which demonstrates that lycorine has an effective antiviral spectrum against ev strains prevalent in mainland china. figure lycorine reduced the virus distribution and the pathological damages. the infected mice were treated with placebo or lycorine at a dose of . mg/kg. the muscle tissues were sampled at dpi, dpi, dpi and dpi for pathological analysis. a, the virus distribution was detected by immunohistological staining. b, the pathological changes were observed after h&e staining. the red arrows indicated virions in tissues, the green arrows for moderate inflammation and the blue for necrotising myositis. magnification: ×. in summary, we have shown that lycorine is a promising drug candidate for treating ev infections that has potential application in the clinical therapy of ev infected patients and that may contribute to the control of ev epidemics in asia. an apparently new enterovirus isolated from patients with disease of the central nervous system the self-limited mouse model was established with a moderate dose of mp infection as described in the methods. the infected-mice were treated with placebo or lycorine at a dose of . mg/kg. the muscle tissues were sampled at dpi, dpi, dpi and dpi respectively (n = ). a, the viral replication curve were determine by qrt-pcr (*: p < . ). b, the viral burden was confirmed by semi-quantitative rt-pcr. c, the clinical scores of the infected mice treated with placebo or lycorine were recorded (n = ). *: p < . . d, photos were a typical transient phonotype of ruffled hair and hind limb paralysis caused by a moderate dose of ev infection at dpi (left enterovirus in taiwan deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. for the outbreak study group neurodevelopment and cognition in children after enterovirus infection hand, foot and mouth disease complicated with central nervous system involvement in taiwan in - clinical features and risk factors of pulmonary oedema after enterovirus- -related hand, foot, and mouth disease molecular epidemiology of human enterovirus in the united kingdom from to enterovirus : epidemiology and diagnostics the enterovirus epidemic in -public health implications for hong kong epidemiologic features of kawasaki disease in taiwan genetic diversity of enterovirus associated with hand, foot and mouth disease epidemics in japan from an outbreak of hand, foot, and mouth disease associated with subgenotype c of human enterovirus in shandong lactoferrin inhibits enterovirus infection by binding to vp protein and host cells ribavirin reduces mortality in enterovirus -infected mice by decreasing viral replication rna interference against enterovirus infection type i interferons protect mice against enterovirus infection toxic amaryllidaceas lycorine and its derivatives for anticancer drug design estevez-braun a: synthesis and antiplasmodial activity of lycorine derivatives suppressive activity of lycoricidinol (narciclasine) against cytotoxicity of neutrophil-derived calprotectin, and its suppressive effect on rat adjuvant arthritis model dose-dependent emetic effects of the amaryllidaceous alkaloid lycorine in beagle dogs plant antiviral agents. iv. influence of lycorine on growth pattern of three animal viruses alkaloids from leucojum vernum and antiretroviral activity of amaryllidaceae alkaloids identification of natural compounds with antiviral activities against sars-associated coronavirus combined peptides of human enterovirus protect against virus infection in mice a simple method of estimating fifty percent endpoints a novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein yeast ribosomal sensitivity and resistance to the amaryllida alkaloids inhibitors of protein synthesis in eukarytic cells. comparative effects of some amaryllidaceae alkaloids on the mechanism of action of alkaloid lycorine characterization and assembly of poliovirus-related s particles complete nucleotide sequence of enterovirus is distinct from poliovirus biochemical characterization of enterovirus d rna polymerase toxidromes associated with the most common plant ingestions lycorine reduces mortality of human enterovirus -infected mice by inhibiting virus replication authors' contributions jl and yy carried out all experiments except for the pathology analysis and draft the manuscript. yx and cm carried out pathology analysis and immunohistochemical staining. cq participated in the designing of experiment and edited the manuscript. lz provided overall supervision, financial support and prepared the final version of the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - ca e wm authors: malhotra, bharti; swamy, m. anjaneya; reddy, p. v. janardhan; kumar, neeraj; tiwari, jitendra kumar title: evaluation of custom multiplex real - time rt - pcr in comparison to fast - track diagnostics respiratory pathogens kit for detection of multiple respiratory viruses date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: ca e wm background: severe acute respiratory infections in children can be fatal, rapid identification of the causative agent and timely treatment can be life saving. multiplex real time rt-pcr helps in simultaneous detection of multiple viruses saving cost, time and labour. commercially available multiplex real time rt-pcr kits are very expensive. therefore the aim of the present study was to develop a cost effective multiplex real time rt-pcr for the detection of respiratory viruses and compare it with an in-vitro diagnostics approved fast track diagnostic respiratory pathogens kit (ftd). methods: nasopharyngeal aspirates and throat swabs were collected and processed for extraction of nucleic acid using an automated extraction system and multiplex real time rt-pcr was performed using the ftd kit and a custom assay on samples. results: custom and ftd assays detected one or more respiratory viruses in ( . %) and ( . %) samples respectively. the concordance between the custom assay and the ftd assay was % for hcov oc , hcov e, hpiv- , hpiv- , hbov, hpev, flu a, and influenza a(h n )pdm and . – . % for the remaining viruses; flu b ( . %), hrv ( . %), hpiv- ( . %), hpiv- ( . %), hcov nl ( . %), hmpv a/b ( . %), rsv a/b ( . %), ev ( . %), hcov hku ( . %), hadv ( . %). major discrepancy was observed for rsv a/b, which was over detected in samples by the custom assay as compared to the ftd assay. the custom assay was much cheaper than the ftd assay and the time taken was only min more. conclusion: the custom primer and probe mix was found to be comparable to the ftd assay with good concordance but was much cheaper and the time taken for reporting was only min more. the low cost custom multiplex rt-pcr can be a useful alternative to the costly ftd kit for rapid identification of viral aetiology in resource limited settings. severe acute respiratory infections (sari) are one of the major causes of illness and death worldwide and are the third most common cause of death among children [ ] . acute respiratory infections (ari) cause more deaths in children < years with most cases reported from india ( million), china ( million), pakistan ( million), bangladesh, indonesia and nigeria ( million) [ ] . respiratory infections can be caused by many viruses, both dna and rna. these include the respiratory syncytial virus (rsv), human parainfluenza virus (hpiv), influenza a virus (flu a), influenza b virus (flu b), human adenovirus (hadv), human coronavirus (hcov), human rhinovirus (hrv), human metapneumovirus (hmpv) and human bocavirus (hbov) [ ] . a new wave of viral diagnosis was established with the development of polymerase chain reaction (pcr) techniques in the s [ ] . pcr is more sensitive and rapid than conventional methods for detection of respiratory viruses. different respiratory viruses present with similar signs and symptoms and can't be differentiated symptomatically or clinically. tests capable of rapid simultaneous identification of various viruses at the same time can help expedite initiation of appropriate therapy. uniplex rt-pcr requires individual amplification of each virus under study which is expensive, time consuming and laborious [ ] . to overcome this, multiplex real-time pcrs targeting the detection of multiple pathogens simultaneously have been developed commercially but they are very expensive. there is a need to develop cheaper systems for rapid simultaneous identification of various viruses. the present study compares custom real-time multiplex pcr primers and probes for the simultaneous detection of respiratory viruses with an in-vitro diagnostics (ivd) approved fast track diagnostics (ftd) kit. children with sari, admitted in j. k. lone hospital, a pediatric hospital attached to sawai man singh (sms) medical college jaipur were enrolled in the study and tested for respiratory viruses with prior consent of the parent/guardian. duration of the study was months i.e. between september, to december, . children enrolled were ≤ years of age, presenting with fever, cough, sore throat, nasal catarrh, shortness of breath, bronchiolitis, pneumonia, and wheezing. samples were not collected from patients with chronic respiratory ailments; non-consenting caregivers, with history of hospitalization in the preceding days, not admitted in hospital and children aged > years. a total of nasopharyngeal aspirate and throat swab samples were collected from patients with sari by a trained technician using a sterile nylon flocked swab and placed in viral transport medium (vtm), labelled and transported on ice at the earliest to advanced research lab (icmr grade- virology lab) of sms medical college jaipur for further processing and storage of the samples. the study was approved by the institutional ethics committee. viral nucleic acid from samples was extracted using an easymag (biomeurex) automated extractor according to the manufacturer's instructions. briefly, the extraction was done from μl homogenised sample which was added to μl lysis buffer and was incubated for min off board. the samples were loaded into the easymag and μl of magnetic silica was added to each sample and mixed well. finally, the nucleic acid was eluted in a volume of μl of which μl was used for the ftd assay and μl for the custom assay. (table ) , μl of extracted nucleic acid of the positive control/sample and made to a final volume of μl with nuclease free water. the thermal cycling profile for the uniplex rt-pcr was °c for min ( cycle), °c for min ( cycle) followed by °c for s and °c for s ( cycles). specimens were considered positive when the ct value was < . uniplex pcr was followed by multiplex real-time rt-pcr standardisation. in multiplex pcr, each reaction . ftd positive samples were considered as standard. agpath (ambion) one step rt-pcr master mix was used for the detection of respective viral nucleic acids as mentioned above in a reaction volume of μl but with μl of nucleic acid, picomoles of each primer and picomoles of probe. the thermal profile for the multiplex real-time pcr was as described above. specimens were considered positive when the ct value was < . after standardisation of multiplex pcr the same protocol was used for screening patient samples. rnase p was used as an internal control in a separate uniplex rt pcr assay. samples negative for rnase p were not included in the study. a total of samples were tested by both assays. custom and ftd assays detected one or more respiratory viruses in ( . %) and ( . %) samples respectively (table ) . no significant differences were seen in the number of samples positive for each virus by the custom assay as compared to the ftd assay except with rsv a/b which was over detected in samples and one sample being under detected by the custom assay as compared to the ftd assay. further, to completely assess the results of these discordant rsva/b samples, testing was repeated using rsv a and rsv b specific primer and probe mix in uniplex real time rt-pcr as published previously [ ] . all samples were found to be positive for rsv b (table ) . one hundred percent concordance was observed between the custom assay and the ftd assay for eight viruses; hcov oc , hcov e, hpiv- , hpiv- , hbov, hpev, flu a, and influenza a(h n )pdm while it varied from . to . % for the remaining ten viruses; flu b, hrv, hpiv- , hpiv- , hcov nl , hmpv a/b, rsv a/b, ev, hcov hku , hadv. (table ). low concordance was observed between the two assays for rsv a/b ( . %) and ev ( . %). the discordant results of the custom assay were seen in co-infection samples, single infection samples and four negative samples as compared to the ftd assay, and the discordance was predominant in the co-infected samples as compared to single infection samples (table ) . comparisons between the custom assay and the ftd assay were made based on the different parameters listed in table . most of the findings between the custom assay and the ftd assay were similar except for the cost incurred for screening respiratory viruses. in this regard, the custom assay was found to be more economical than the commercial ftd assay. the present study was performed to compare a custom multiplex assay and an ftd multiplex assay by testing of respiratory samples obtained from children with sari admitted in j k lone paediatric hospital jaipur. in the present study, the concordance between the custom assay and the ftd assay was found to be % for flu a, influenza a(h n ) pdm , hcov oc , hcov e, hpiv- , hpiv- , hbov, and hpev. similarly chen et al., [ ] reported a concordance of . % for flu a and influenza a(h n ) pdm when comparing a multiplex pcr assay with a uniplex assay. the concordance between the two assays varied from . to . % for the remaining ten viruses; [ , ] . concordance for ev in the present study was different from an earlier study ( . %) [ ] . the difference in concordance obtained in different studies may be due to the different primer binding regions or may be due to different methodologies employed by various studies. the number of samples positive for hcov e, hpiv- , hpiv- , hcov nl , hpev, hcov hku , flu a, were ≤ in the present study. studies based on larger numbers of samples are required to assess the concordance of these viruses more thoroughly. the limit of detection for some of the viruses in the custom assay (table ) ranged from dna copy/ml to × copies/ml [ , [ ] [ ] [ ] [ ] [ ] . the detection limit of the ftd assay for different viruses was copies/ml for flua, hpiv- , hmpv and hcov oc ; copies/ml for flub, hcov hku , hpiv- , hbov, hpiv- , hcov nl , rsv, hadv, ev, and hpev; and copies/ml for hrv, hcov e and hpiv- [ ] . in the present study rsv a/b was the most predominant virus detected by both the custom and ftd assays with positivity in ( . %) and ( . %) samples respectively and concordance of . %. this finding is different when compared with other studies [ , ] where comparisons were made between multiplex pcrs in which rsv was the second most predominant virus detected [ ] . the custom primer and probes used for influenza a (h n ) [ ] , and . % [ ] respectively in earlier studies where the same primer and probes were used. hbov was mostly associated with co-infections in the present study in both assays. this is consistent with an earlier study [ ] the major discrepancy in the present study was found with rsv a/b. the discrepancy in samples which were over detected by the custom assay was resolved by rsv a and rsv b typing. the rsv typing results for the discrepant samples showed that all samples were rsv b. further all samples positive for rsv a/b by the ftd assay were also subjected to rsv typing which indicated rsv a in ( . during the process of standardisation of the custom assay μl of viral nucleic acid (positive control) was used for each virus including picomoles of primers and picomoles of probes. each panel consisted of viruses. in total μl of viral nucleic acid was used for each panel. while the ftd assay used ul of nucleic acid in each tube with primers and probes for viruses, the concentration of primer and probe was not disclosed by ftd. in total μl more of viral nucleic acid was used in the custom assay compared to the ftd assay which may have increased the sensitivity/detection of different viruses in the custom assay. initially during the process of standardisation of the custom assay, different primer and probe concentrations were tried and the pcr was run for cycles as per the protocol followed by various authors. although data was analysed using pcrs run for and cycles, best results were achieved using a ct value of for both the ftd assay and the custom assay. accordingly, a ct value of < was considered as positive for both assays as per the ftd kit. with the custom assay being run for cycles this reduces the custom assay run time by min, thereby making it only min longer than the ftd assay. comparisons were made between various aspects of the custom and the ftd assays (table ). no major differences were observed between the two assays except in the cost incurred for both assays. similar comparisons were also done in an earlier study [ ] where three multiplex pcrs were compared. the turn-around time of the custom assay was min more as compared to the ftd assay. but both the assays reported the results on the same day. the excess time of min taken by the custom assay as compared to the ftd assay may not greatly interfere with the treatment process. however, the custom assay was much more economical costing inr /-per sample for screening respiratory viruses compared to the commercial ftd assay which was expensive costing inr /-per sample. this assay may prove to be highly cost effective in resource limited settings like ours. however the limitation of our study was that some of the viruses showed low positivity as a result it is difficult to assess the concordance accurately. larger numbers of positive samples need to be tested to evaluate the concordance of these less prevalent viruses. this study reported a high prevalence of respiratory viruses in children ≤ years using a custom assay and an ftd assay. good concordance was observed for all the viruses between both assays except for rsva/b. however larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses. human bocavirus infection among children estimates of worldwide distribution of child deaths from acute respiratory infections acute respiratory viral infections in children in rio de janeiro and teresopolis new respiratory viruses and the elderly respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection simultaneous detection of influenza a, influenza b, and respiratory syncytial viruses and subtyping of influenza a h n virus and h n ( ) virus by multiplex real time pcr comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana human coronaviruses hcov-nl and hcov-hku in hospitalized children with acute respiratory infections in beijing, china detection of all known parechoviruses by real-time pcr? clinical evaluation of multiplex real-time pcr panels for rapid detection of respiratory viral infections molecular assays for quantitative and qualitative detection of influenza virus and oseltamivir resistance mutations validation -ftd respiratory pathogens . luxembourg: fast track diagnostics evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood viral aetiology of acute lower respiratory tract illness in hospitalized paediatric patients of a tertiary hospital: one year prospective study real-time pcr assays for detection of bocavirus in human specimens during the summer outbreak of "swine flu" in scotland what respiratory pathogens were diagnosed as h n / ? comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder assay and respifinder smart assay cdc protocol of real-time rtpcr for influenza a (h n ) replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time pcrs authors acknowledge the financial support from indian council of medical research to bm for setting up icmr grade-i viral research and diagnostic laboratory and senior research fellowship to mas. the custom primer and probe mix was much more economical than the commercial ftd kit. our study suggests that this custom multiplex real-time rt-pcr can be used for simultaneous and rapid detection of multiple viruses in resource limited settings. this will help prevent unnecessary use of antibiotics and permit timely initiation of supportive therapy/antiviral drugs if available. authors' contributions bm participated in conception and design, experimental studies, analysis and interpretation of data, drafting the manuscript and revising it critically for important intellectual content, final approval of the version to be published, and agree to be accountable for all aspects of the work. mas participated in conception and design, experimental studies, acquisition of data, analysis and interpretation of data; drafting the manuscript and revising it critically for important intellectual content, final approval of the version to be published. pvjr participated in conception and design, experimental studies, analysis and interpretation of data; drafting the manuscript and revising it critically for important intellectual content, final approval of the version to be published. nk participated in conception and design, drafting the manuscript and revising it critically for important intellectual content, final approval of the version to be published. jkt participated in conception and design, drafting the manuscript and revising it critically for important intellectual content, final approval of the version to be published. the authors declare that they have no competing interests. key: cord- -dhwlr ui authors: lednicky, john a; waltzek, thomas b; mcgeehan, elizabeth; loeb, julia c; hamilton, sara b; luetke, maya c title: isolation and genetic characterization of human coronavirus nl in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: dhwlr ui background: cryopreserved primary human renal proximal tubule epithelial cells (rptec) were obtained from a commercial supplier for studies of simian virus (sv ). within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the rptec. the rptec henceforth deteriorated rapidly. since sv induces the formation of cytoplasmic vacuoles, this batch of rptec was rejected for the sv study. nevertheless, we sought the likely cause(s) of the deterioration of the rptec as part of our technology development efforts. methods: adventitious viruses in the rptec were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, pcr, and sequencing. results: cytomegalovirus (cmv) was detected in some rptec by cytology, an immunofluorescence assay, and pcr. human herpesvirus b was detected by pcr of dna extracted from the rptec, but was not isolated. human coronavirus nl was isolated and identified by rt-pcr and sequencing, and its replication in a fresh batch of rptec and another type of primary human kidney cells was confirmed. conclusions: at least different adventitious viruses were present in the batch of contaminated rptec. whereas we are unable to determine whether the original rptec were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with hcov-nl from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. importantly, hcov-nl replicates to high titers in some primary human kidney cells. cell lines and primary cells obtained from commercial suppliers or through inter-laboratory transfer can contain adventitious (i.e., contaminating) viruses. this happens primarily because cytopathic effects (cpe) are not always apparent in virus-infected cell cultures, and consequently, the cells are unwittingly sold or transferred between laboratories [ ] . the adventitious viruses that are encountered in cell cultures often stem from bovine serum that is used to supplement cell growth media, and include: bovine viral diarrhea virus (bvdv) [ ] [ ] [ ] [ ] [ ] [ ] , bovine polyomavirus [ , , ] , bovine parvovirus [ , [ ] [ ] [ ] (j. lednicky, unpublished) , and bovine herpes viruses [ , [ ] [ ] [ ] [ ] . unintentional contamination of cultured cells by these serum-derived viruses has obvious consequences not only with regard to data generation, but also because it exerts a toll on time wasted in the performance of laboratory work, and the costs thereof. other common sources of contaminating viruses are: (a) laboratory workers, and (b) animal-sourced enzymes (such as porcine trypsin) and (c) other biologicals that are used for cell culture [ ] . examples of viruses that stem from porcine trypsin that have recently been found as contaminants of many cell lines including those used for vaccine production are torque teno sus virus (ttsuv), a member of the family anelloviridae, and porcine circoviruses and (pcv and pcv ) [ , [ ] [ ] [ ] [ ] [ ] . anelloviruses and circoviruses are relatively small viruses with single-stranded, circular dna genomes that replicate within the nuclei of infected cells. cpe due to the presence of anelloviruses have not been well described at present. finally, primary cells can contain endogenous retroviruses and other viruses. for example, primary monkey kidney cells, which are used for the detection of paramyxoviruses and picornaviruses in many american diagnostic microbiology laboratories, can contain endogenous simian viruses that are either latent in the kidneys, or cause persistent but inapparent kidney infections in their hosts [ ] . the work described in this manuscript resulted from a previous study of sv transcription in primate cells (j. lednicky, unpublished) . sv is a polyomavirus that was once referred to as "vacuolating agent" or "simian vacuolating virus " because commonly studied sv strains induce the formation of cytoplasmic vacuoles late during infection of most permissive primate cells [ ] . a batch of primary human rptec that had been obtained for our previous transcription study of well-known vacuolating strains of sv proved unsuitable, as about % of the cells exhibited cytoplasmic vacuolation within hours after they were seeded in flasks. necrosis and apoptosis were also evident in some of the attached cells. due to vacuolation and obvious cell deterioration, the rptec were rejected for our sv study. nevertheless, as we often work with primary cells and continuously refine our research methodologies, we sought to determine a likely root cause(s) of the deterioration of the rptec to (a) advance our understanding of primary cell culture technology, and (b) explore whether proper biosafety practices were being observed. for example, might the rptec be contaminated with a significant pathogen best suited for work in biosafety level- or − laboratories? we first tested whether vacuolation of the rptec stemmed from faulty media preparation. for example, vacuoles can form in madin darby canine kidney (mdck) cells due to: (a) shortage of l-glutamine in the cell growth medium, (b) inappropriate addition of antifungal agents to the medium, (c) improper co environment for the sodium bicarbonate concentration of the medium, (d) nutrient depletion of the medium, and (e) mycoplasma contamination [ ] . faulty media formulation was ruled out as the root cause of the failure of this batch of rptec to thrive. instead, based on the progressive formation of cpe, the results of our initial diagnostic tests, and our cumulative experience with cell culture [ ] , we predicted that adventitious agents were causing the rapid demise of our rptec cultures. dna extracted from the rptec tested negative by pcr for mycoplasma species, and polyomaviruses sv and bk virus (bkv), suggesting none of these was causing vacuolation and/or cell deterioration. however, a single cause of the rptec deterioration was unlikely, as we detected different human viruses in the rptec: human cytomegalovirus (cmv), human coronavirus nl (hcov-nl ), and human herpesvirus b (hhv- b). cmv, also known as human herpesvirus- (hhv- ), (subfamily betaherpesvirinae), is a double-stranded dna virus that establishes lifelong persistence; it can remain latent in different human tissues and is known to infect renal tubular epithelial cells. a majority of humans are seropositive for cmv [ , ] . whereas cmv infections are typically asymptomatic in healthy humans, the virus can reactivate and cause disease in immunosuppressed patients, including those undergoing kidney transplantation. indeed, cmv antigens and dna are found in renal epithelial cells in kidneys of trauma victims examined during autopsy as well as in biopsies of renal allografts, indicating that these cells can harbor cmv in both healthy persons and allograft recipients [ , ] . hcov-nl is a single-stranded positive-sense rna virus of the genus alphacoronavirus (family coronaviridae, order nidovirales). first identified in from a child with bronchiolitis in the netherlands, it is now recognized that hcov-nl can cause upper and lower respiratory tract infections in humans, primarily in infants and the elderly [ ] [ ] [ ] [ ] [ ] [ ] . wild-type hcov-nl is difficult or impossible to isolate from clinical specimens in continuous cell lines [ ] , although the prototype hcov-nl strain was propagated in llc-mk cells [ ] and in primary, differentiated human bronchial-tracheal respiratory epithelial cells cultured at the air-liquid interface [ ] . there are at least three different hcov-nl genotypes (a, b, and c) [ ] . hhv- b is a double stranded dna virus (subfamily betaherpesvirinae, genus roseolovirus) that infects up to % of humans and is the causative agent of exanthem subitum, which is also known as roseola infantum or sixth disease [ ] . after the primary infection, hhv- b generally persists in latent form in t-lymphocytes and other cells. hhv- b reactivation is common in transplant recipients, which can cause several clinical manifestations such as encephalitis, bone marrow suppression and pneumonitis [ ] . the work presented herein serves as a reminder that biologicals (such as calf serum and cultured cells) can be contaminated with adventitious agents. the focus of this article is on the detection and isolation of hcov-nl , which to our knowledge, heretofore has not been reported in a natural infection of human kidney cells, or tested in vitro in primary human rptec. within hrs after cryopreserved rptec were thawed and seeded in cell culture flasks, we observed that about % of the attached cells were vacuolated. since vacuolation may have been a sign of cytotoxicity due to residual cryopreservative, the rptec basal growth medium [basal growth medium (bgm)], which had been supplied with the cells, was changed. we noted by phase-contrast microscopy that prominent intranuclear inclusions surrounded by a clear halo ("owl-eyes") were present in enlarged nuclei in some of the rptec, and that the same cells were enlarged relative to a majority of the others. these findings were considered pathognomonic for cytomegalovirus (cmv) [ ] (table ) . vacuoles were still present hrs post-seeding of the rptec (and after the rgm change at hrs) ( figure a ), but there were no signs of contamination by extracellular bacteria or fungi. the ph at °c of fresh bgm was approximately . (within normal range), and ammonia was not detected using a salicylate-based method (data not shown). these findings suggested neither incorrect ph nor presence of ammonia in bgm were causing vacuolation of the rptec. moreover, cv- , llc-mk , and vero cells, which are cell lines derived from monkey kidneys, did not get vacuolated after hrs incubation with bgm. thus, no evidence of cytotoxicity due to bgm was uncovered. by hrs post-seed, vacuoles were still present in rptec in bgm that had been boosted with additional l-glutamine, suggesting glutamine deficiency was not an issue. a bioactive agent release assay indicated something in the spent bgm of the hr rptec cultures induced enlargement and/or vacuolation of wi- ( figure b and c), llc-mk (figures a and b) , vero e cells ( figure c ), and cv- and hek- cells (not shown) within hrs. cell enlargement, rounding, and vacuolation were more notable in wi- cells than other cells (table ). these observations suggested the rptec were releasing either a biomolecule(s) or virus(es) that adversely affected some of the cell lines. immunofluorescence assay (ifa) and pcr for cmv some rptec from hr cultures were positive for cmv by ifa (their nuclei were fluorescent), and dna extracted and purified from the cells also tested positive for cmv by pcr (data not shown). however, the extracted dna was pcr negative for human herpes virus (hhv)- and hhv- , and polyomaviruses sv and bkv ( table ) . cpe consisting of cell swelling/rounding and/or vacuoles also occurred at °and °c in wi- , cv- , llc-mk , and vero cells inoculated with spent media from -day old rptec cultures. as for the bioagent release assay, morphological aberrations were most notable in wi- cells. trypsin did not enhance cpe in llc-mk , mdck, mdck-london, mv lu, or vero cells. the wi- cells (but not the other cells) died days afterwards. however, starting day post-inoculation (p.i.), occasional syncytia with or more nuclei were noted in llc-mk , cv- , hek- , mv lu, and vero cells, and smaller syncytia (with up to nuclei) in mdck and mdck-london cells (table ) . thereafter, cpe were most pronounced in llc-mk cells and in hek cells. in llc-mk cells, most of the early cpe consisted of vacuolation and the formation of foci of detached rounded cells, many forming elongated oblong clumps of rounded cells above the monolayer (referred to as "striations" in ref. [ ] ). at later times, cytolysis of syncytia occurred. vacuolation in llc-mk cells appeared more pronounced at °than °c, and conversely, syncytia appeared larger at °than °c ( figures a-d) . rounding followed by eventual detachment from the growing surface occurred in infected hek- cells (not shown). in mdck cells, vacuoles were also more pronounced at °than °c. due to cell vacuolation and deterioration, electron micrographs of five day old rptec cultures were difficult to interpret. at low magnification, vacuoles and cell deterioration were obvious ( figure a ). occasional viral particles consistent in appearance and size with cmv at different stages of maturation were observed at higher magnifications (data not shown). in addition to nuclear inclusions, homogenous electron-opaque, dense cytoplasmic bodies were present. however, unlike the irregular-shaped cytoplasmic bodies we usually observe in cmv-infected cell cultures (j. lednicky, unpublished observations), these were distinctly circular, as described by smith and de harven for cmv in infected cells [ ] ( figure b ). additionally, we also noted many virus-like particles (vlp) that were morphologically different from cmv; these vlp were present as free particles, within vacuoles, and in transport vesicles. a majority of the virus-like particles were spherical, and they collectively ranged from about to nm in diameter, and some seemed to have surface projections ( figure c ). somewhat different results were obtained when the indicator cells of table were inoculated with freeze-thawed rptec lysate from -day old cultures instead of spent media from -day old rptec cultures. in contrast to previous findings, cpe were not observed in wi- cells at early times onto days p.i. however, cpe were seen in llc-mk cells starting days p.i., and in other cells at later times ( table ) . as before, vacuolation was more pronounced at °than °c. since syncytia were observed, we focused pcr efforts on the detection of the viruses that we considered the most likely candidates: coronaviruses, human paramyxoviruses, and reoviruses (hhv- and − were already ruled out, section , above). we did not test for retroviruses, acknowledging that exogenous or endogenous retroviruses may have been causing syncytia in the cells. extracted nucleic acids were tested by pcr or rt-pcr using assays designed to detect known human coronaviruses [ , [ ] [ ] [ ] , paramyxoviruses [ ] [ ] [ ] , and reoviruses [ ] . rt-pcr and sequencing showed one of the viruses in the cv- , hek- , llc-mk , mdck, mdck-london, mv lu, and vero e cells was coronavirus hcov-nl . an example of rt-pcr reactions performed with primer sets specific for hcov-nl is shown in figure . proof that hcov-nl was replicating in the llc-mk cells was obtained by electron microscopy ( figure a -e). characteristic features of hcov-nl replication in llc-mk cells [ , ] were detected, such as the formation of double membrane and laminar structures, and inclusion bodies ( figure a ). packets of granular nucleocapsid material were also evident in infected cells ( b). virus particles at various stages of maturation were present in the cytoplasm ( c) and in the rer outside the nuclei ( d). free virus particles - nm in diameter were present in spent media ( e). a counterstain was not used to easily visualize the viral spikes ("crown") surrounding the viruses in figure e . the complete consensus genomic sequence of hcov-nl was obtained for virus in llc-mk cells that had been incubated at °c. the virus, designated hcov-nl strain rptec/ / , has a genomic length of , bp, and the complete sequence has been deposited in genbank (accession no. jx ). a dataset was prepared containing complete or nearly complete hcov-nl genomes in genbank. to construct a phylogram, the for more comprehensive analyses. pcr tests for herpesviruses that were not included in our previous assays (for hhv- ,- ,- ,- , and − ) were performed on dna extracted from rptec. a -bp amplicon was generated using nested primers for hhv- [ ] . identity was confirmed by sequencing (data not shown). biotypes of plaque-purified hcov-nl /rptec/ compared to hcov-nl /amsterdam- since it was likely that multiple viruses contributed to the observations described in table , an attempt was made to plaque purify hcov-nl in caco- cells [ ] at °c [ , ] and in llc-mk cells at °c ( °to °c are considered optimal temperatures for the in-vitro cultivation of hcov-nl [ ] [ ] [ ] , , ] ). whereas hcov-nl replicates more effectively in caco- cells than llc-mk cells [ ] , that information was not available and therefore caco- cells were not used in our initial studies (table ) , which were performed in . nine days p.i., llc-mk cells were stained with neutral red, individual plaques picked, and subjected to more round of plaque purification [ ] . similarly, foci of cpe were identified under an unstained agarose overlay in caco- cells days p.i., picked, and subjected to more rounds of plaque purification [ ] . plaque-purified stocks resulting from llc-mk figure rt-pcr detection of hcov-nl in llc-mk cells. lane m, bp mw markers (new england biolabs); lane , hcov-nl specific pcr product ( bp) amplified by pcr primers n -pcr and n -pcr [ ] ; lane , hcov-nl -specific pcr product ( bp) amplified by pcr primers repsz- and sz- [ ] ; lane , non-infected llc-mk control tested using pcr primers n -pcr and n -pcr ; lane , non-infected llc-mk control tested using pcr primers repsz- and sz- . table were infected at a moi of . pfu/cell. hcov-nl /rptec/ pp a -f) formed the same cpe described in table that were observed for freeze-thawed rptec, though formation of cpe was delayed by at least day. a few examples are depicted in figure a cells. cpe were least obvious in mdck and mv lu cells. with the exception of hek- cells, which were only tested at °c (below °c, these cells do not adhere well to the growing surface of a flask), cpe were first detected at °c. from spent media harvested from -day old cultures, viral titers were obtained for hcov-nl /rptec/ pp isolates a -f and hcov-nl -amsterdam- using plaque assays in caco- cells [ ] . for each cell line that was tested (as listed in table ), the viral titer was similar for each virus. representative results, obtained for hcov-nl /rptec/ pp isolate a ( figure a ), indicate the highest titer ( . × pfu/ml) was attained when the virus was propagated in llc-mk cells. using a moi of . pfu/cell, we tested progeny virus production by hcov-nl /rptec/ pp a and d in llc-mk cells. the virus yields over a -day infection period were determined by plaque assays in caco- cells. similar results were obtained for the viruses; the results for rptec/ pp d are shown in figure b . newly acquired (in ) primary rptec, hre, and hrce cells did not release a detectable bioagent (data not shown). what may have been "owl's eye" nuclei were observed rarely only in hre cells. both hcov-nl /rptec/ and hcov-nl /amsterdam- caused rapid formation of cpe in rptec ( figure ) and hre cells (figure ) infected at a moi of . with plaque purified hcov-nl /rptec/ pp a or hcov-nl /amsterdam- . we noted that the rptec were not vacuolated when sub-confluent ( figure a ) yet became vacuolated once confluent ( figure b ), but otherwise stayed viable when re-fed every days with rebm. extensive cpe consisting of rounding of the cells and cytolysis occurred by dpi in rptec ( figure c -e) and dpi in hre cells ( figure b -c). when ml of spent rebm was obtained from rptec or hre cells days after they had been infected with hcov-nl rptec/ pp a or hcov-nl /amsterdam- , and inoculated onto llc-mk cells in t flasks, cpe were extensive days later ( figure f and figure d ). in contrast, ml of spent media from non-infected (negative control) rptec and hre cells had no effect on llc-mk cells (data not shown). the presence of hcov-nl in the spent media of rptec and hre that had been inoculated with the viruses, and in the indicator llc-mk that had been inoculated with spent media from the virus-infected cells, was confirmed by rt-pcr (data not shown). in contrast, cpe were sparse in hrce cells dpi with either hcov-nl /rptec/ pp a or hcov-nl /amsterdam- (data not shown). both hcov-nl /rptec/ and hcov-nl / amsterdam- formed relatively high viral titers by dpi in rptec and hre cells, but not in hrce cells ( figure ). the viral titers exceeded those formed in llc-mk cells by about orders of magnitudes (ie, by logs). in contrast, viral titers for both virus strains remained low ( pfu/ml) in hrce cells by dpi (data not shown). the presence of cmv in the original batch of viruscontaminated rptec was not a surprise to us, as we have isolated cmv from frozen (− °c) simian kidneys and from primary simian kidney cells (lednicky, unpublished) . we learned from the supplier that the donor of the virus-contaminated rptec of this study was seropositive for cmv. however, our batch of viruscontaminated rptec was not checked for the presence of cmv by the supplier (personal communication). as precedence for the presence of cmv in human kidney cells in vivo, it is known that reactivation of cmv in renal tubule epithelial cells can complicate kidney transplantation, leading to poor long-term graft function [ ] . the apparent complete inactivation of cmv by the freeze-thaw procedure we used was unexpected, as the process does not always completely inactivate cmv [ ] , but was nevertheless fortuitous, leading to observations resulting in the detection of hcov-nl . then again, it may have inactivated other viruses in the rptec. to our knowledge, ours is the first description of hcov-nl in primary rptec. overall, our observations of hcov-nl growth in various cell lines appear consistent with literature reports. growth of the virus in llc-mk and vero cells is well known [ , , ] . the ability of the virus to form cpe in mdck was previously described [ ] . the lack of hcov-nl growth in human fibroblasts has been reported [ ] . in particular, mrc- cells, did not support the replication of hcov-nl [ ] , and those cells are used interchangeably with wi- cells in american diagnostic virology laboratories for the isolation of respiratory viruses and cmv (both cell lines are derived from human fetal lung cells). thus, it is not surprising that hcov-nl does not replicate in wi- cells. growth of hcov-nl at °c has been reported and should not be a surprise [ , ] . that hcov-nl might induce vacuolation is not a surprise, as that is a common property of coronaviruses. it will be interesting to see if interaction with ganglioside gm is related to the vacuolation process, as reported for sv [ ] . hcov-nl replicated in hek- cells, as does sars-cov [ , ] . both sars-cov and hcov-nl can use angiotensin-converting enzyme (ace ) as a viral receptor [ ] , and ace is expressed in kidneys [ ] , and may be reasons hcov-nl was present in our batch of rptec and could infect hek- cells. replication of sars-cov in mv lu cells was previously reported [ ] , so perhaps it is not surprising that hcov-nl does as well, if the viruses share receptor specificity, and mv lu cells contain the cellular machinery necessary for the replication of these viruses. however, the origin of hek- is unclear, as the cells express neurofilament (nf) subunits nf-h, nf-l, nf-m, alphainternexin, and other proteins found in neurons [ ] . thus, hek- may be of neuronal origin, and it will be interesting in the future to discern which neural and kidney cells support the replication of hcov-nl . it is not clear why rapid cell swelling rounding, and vacuolation, followed by cell death, occurred in wi- cells. our current hypothesis is that cmv was latent in the kidney cells of the donor of the rptec, and that the virus was reactivated during the initial harvest of cells from the donor's kidney. we surmise that within our batch of rptec, that many of the cells had been inadvertently frozen when they were at an early stage of cmv infection. it is likely that the cells produced a large yield of cmv when they were brought out of cryopreservation, and that the high-titer cmv infected the permissive wi- at a high moi, and this resulted in rapid killing of those cells. since we were unprepared for such analyses, a quantitative enumeration of infectious cmv particles was not performed. we also suspect that cmv from the rptec had infected vero, llc-mk , and cv- cells, but the infection was abortive [ ] , unlike the situation in wi- cells, which are permissive for that virus. finding that the hcov-nl is similar to viruses from and is perhaps not surprising, as the rptec of this report were prepared from a donor and purchased (by us) that same year. to our knowledge, hcov-nl has not been reported in natural infections of human kidneys. the ability of hcov-nl to replicate to high titers in primary rptec and hre cells suggests that at least some human kidney cells are fully permissive for the virus. however, we are unable to resolve whether (a) the original batch of contaminated rptec were infected (naturally) with the virus prior to harvest, or (b) a worker with a respiratory infection accidentally contaminated the rptec during their initial preparation, or (c) the rptec were contaminated in our laboratory. we are unable to resolve the issue whether the cells were contaminated during preparation for many reasons, foremost being the company that sold the cells was merged with a different entity. it is unlikely that the rptec were infected in our laboratory, as we did not have hcov-nl in our laboratory in , and acquired hcov-nl /amsterdam- only recently (sept. ) so that we could compare the biotype of hcov-nl /rptec with that of amsterdam- . moreover, our laboratory policy dictates that workers refrain from cell culture work when they have a respiratory tract infection. it is plausible (but we lack proof) that hcov-nl may have been latent in the donor's kidneys, a possibility consistent with the known biology of various coronaviruses that establish long-term but sub-clinical infections. noteworthy, sars-cov, which shares the same ace receptor as hcov-nl , has been associated with kidney disease [ ] [ ] [ ] [ ] . sars-cov causes a systemic infection with viral shedding not only in respiratory secretions, but also in stool and urine [ , , ] . perhaps hcov-nl is capable of causing systemic infections as well, though the severity is much less than that of sars-cov. a parallel to this notion is the finding that hcov-nl replicates to high titers in caco- cells [ ] , which are derived from a human colon carcinoma. in april of , a new coronavirus capable of causing severe acute respiratory infections of humans emerged in jordan. the same coronavirus was isolated in the summer of from a patient with acute pneumonia and renal failure in saudi arabia [ , ] . the new virus has been fully sequenced, classified as a group c β-coronavirus [ ] [ ] [ ] , and termed middle east respiratory syndrome coronavirus (mers-cov) by the coronavirus study group of the international committee on taxonomy of viruses (announced in j. virology on may , ) . genetically, mers-cov is closely related to sars-cov, and is another example of a coronavirus associated with respiratory disease that can also infect kidney cells. the donor of the rptec of our study did not have kidney disease (otherwise, the cells would not have been harvested and sold for research purposes), suggesting a persistent, sub-clinical infection of the kidneys by hcov-nl is more likely. to what extent, if any, hhv- b may have somehow modulated the growth of the other viruses in the rptec is unclear. noteworthy, hhv- b has also been reported in association with renal epithelial cells and kidney transplant rejection [ ] . lastly, whereas the virus-like particles of figure c appear similar to those in an electron micrograph of sars-cov in kidney tissue [ ] , we have no formal proof that they are in fact hcov-nl and may be another virus we did not identify in our work. taken together, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. moreover, our findings raise the possibility of kidney involvement during the course of infection with hcov-nl . cryopreserved primary human rptec were obtained from a commercial source in the usa. bgm, supplements, and growth factors [fetal bovine serum, insulin, transferrin, triiodothyonine (t ), human recombinant epidermal growth factor, hydrocortisone, epinephrine, gentamicin sulfate, and amphotericin-b] were concurrently obtained as a kit from the rptec supplier. the rptec were first seeded onto four t flasks and manipulated following instructions included with the kit. glutamine, which was later substituted with mm l-alanyl-l-glutamine (glutamax™, invitrogen corp.). both dmem and emem were supplemented with antibiotics [psn; μg/ml penicillin, μg/ml streptomycin, μg/ml neomycin (invitrogen corp.)], and % (v/v) low igg, heat-inactivated gamma-irradiated fetal bovine serum (hyclone, logan, ut). additionally, sodium pyruvate (invitrogen corp.) and non-essential amino acids (hyclone) were added to emem., with the exception: emem formulated with calf serum (hyclone) instead of fbs was used for nih/ t cells. before seed stocks were prepared, the cell lines were propagated in growth media with plasmocin (invivogen, san diego, ca) for weeks to reduce the chances of mycoplasma contamination. next, the cell lines were incubated for a minimum of weeks in the absence of antibiotics to determine whether fast-growing microbial contaminants were present or abnormal morphological changes would occur (associated with intracellular mycoplasma). following - weeks of propagation without antibiotics, the plasmocin-treated cell lines and rptec cells were tested by pcr for the presence of mycoplasma dna using a takara pcr mycoplasma detection kit (fisher scientific, pittsburgh, pa) [ ] . the cells tested negative for mycoplasma. an independent laboratory (at the university of florida) confirmed that the stock of llc-mk cells that was used for the isolation of hcov-nl in this manuscript was negative for human respiratory viruses including human coronaviruses e, hku , oc , and nl using a genmark multiplex respiratory pcr esensor xt- respiratory viral panel (esensor rvp; genmark diagnostics, inc., carlsbad, ca). fresh l-glutamine was added to bgm in a hr rptec culture and the cells observed every six hrs for one day to assess the effect on cell morphology, vacuolation, and viability. complete, freshly prepared bgm was substituted for dmem in subconfluent cultures of cv- , llc-mk , mdck, vero, and wi- cells, and the cells incubated at °c and observed every hours over days for morphological changes or cell death as evidence of cytotoxicity. to find out whether the rptec were releasing a bioactive agent, spent bgm from a hr rptec culture was equally subdivided and added to subconfluent cv- , hek- , llc-mk , vero e , and wi- cells in t- flasks. these particular cell lines were chosen on the assumption that a virus growing in rptec would preferentially infect primate over non-primate cells. after inoculation, the cells were incubated at °c (the same temperature used for rptec) and observed for morphological aberrations over hrs. a standard cytospin procedure was used to deposit rptec from a hr culture onto a glass slide. ifa was performed using a commercial kit with a primary antibody directed against a cmv immediate early protein, and a secondary antibody that was labeled with fluorescein isothiocyanate (light diagnostics™ cmv ifa kit, millipore, billerica, ma). the bgm of a five day rptec culture was replaced with fresh ice-cold cacodylate-buffered % gluteraldehyde (ph . ). after hrs at room temperature, the fixed cells were scraped free using a cell scraper, and pelleted by centrifugation at , x g for minutes. the fixative was removed, and the cell pellet resuspended with cold fixative to a final volume of μl, then stored overnight at °c. the fixed cells were post-fixed with osmium tetroxide, stained with uranyl acetate, embedded in spurr's embedding medium, then thin-sectioned. the thin sections were stained with uranyl acetate and lead citrate and transmission electron microscopy performed using a hitachi h- . five days after being seeded, about % of the rptec had completely deteriorated, whereupon spent bgm media was added to groups of subconfluent a , bhk- , cv- , hek- , llc-mk , mdck, mdck-london, mv lu, nih/ t , vero e , and wi- cells in complete growth media, and to groups of llc-mk and mdck and mv lu cells in serum-free media containing l- -tosylamide- -phenylethyl chloromethyl ketone (tpck)-treated trypsin. the tpck-trypsin was at a final concentration of μg/ml (mdck and mdck-london cells) or . μg/ml (llc-mk and mv lu). for each group, set was incubated at °c, the other at °c (incubation at different temperatures is standard in our laboratory, as many of the respiratory viruses we work with preferentially replicate at temperatures lower than °c). tpck-trypsin in serum-free media was used to facilitate the isolation of influenza and other viruses that require protease cleavage of some viral component for infectivity. after inoculation, the cells were re-fed every days with % serum media or serum-free media with trypsin for long-term (up to day) observations. at day post-seed, only about % of the rptec remained attached to the flask, a majority of which were vacuolated and showed other signs of cpe. to facilitate the isolation of viruses other than cmv, the cells were scraped free and transferred along with the spent bgm into a sterile ml polypropylene centrifuge tube, and frozen at − °c for one week (this step reduces the number of viable cmv virions by a factor of many logs, since cmv loses viability when stored at − °c) [ ] ; [j. lednicky, unpublished] . next, the frozen tube of scraped rptec was freeze-thawed three times, alternating between freezing at − °c for hrs and a minute thaw at room temperature, as an additional measure to further reduce the number of viable cmv particles. after the third thaw, an aliquot was tested using the cells and methods of section . above, and the remainder frozen at − °c for retrospective analyses. intracellular dna was purified from a hr rptec culture using a qiaamp dna mini kit (qiagen, valencia, ca) and tested by pcr for cmv, hhv- and − , and polyomaviruses sv and bkv. total rna was purified from a freeze-thawed seven-day old rptec culture supernatant using a qiaamp viral rna kit (qiagen). the primers and conditions that were used for pcrbased detection of viruses were based on published literature and will be provided upon request. since syncytia were formed by the second virus (not cmv) that we were attempting to identify, pcr efforts were focused on human herpes, paramyxo (measles, mumps, metapneumovirus, parainfluenza viruses - , respiratory syncytial virus), and coronaviruses. rt-pcr for rna virus screens was performed with omniscript reverse transcriptase (qiagen) followed by pcr with hotshot taq (new england biolabs, ipswich, ma) °c. hcov-nl was first detected using a pancoronavirus rt-pcr assay for the viral polymerase gene with primer pair cor-fw and cor-rv [ ] , followed by sequencing of the bp amplicon. that was accomplished using cor-rv for cdna synthesis (with reverse transcription performed for hr at °c), and pcr performed as: initial denaturation step: °c ( . min); cycles of °c ( sec), °c ( sec), °c ( sec); terminal extension step at °c ( . min); °c ∞. for confirmation, primer pairs n -pcr and n -pcr [ ] and repsz- , and repsz- [ ] were used with pcr parameters similar to those for cor-fw and cor-rv, and the resulting amplicons sequenced. n -pcr and n -pcr amplify a bp amplicon from the hcov-nl nucleocapsid region. n -pcr was used to generate cdna, and pcr performed at an annealing temperature of °c. following cdna synthesis primed with repsz-rt [ ] , primer pair repsz- , and repsz- amplify a bp amplicon from the hcov-nl orf b region at a pcr annealing temperature of °c. electron microscopy of llc-mk cells infected with hcov-nl from rptec llc-mk cells that were rt-pcr positive for hcov-nl were trypsinized to detach them from the growing surface of a t flask, pelleted, and the pellet resuspended in icecold % paramormaldehyde, % gluteraldehyde, in . m sodium cacodylate, ph . . they were subsequently analyzed as described above. targeted hcov-nl /rptec/ sequences were rt-pcr-amplified from purified rna using a genome walking strategy. briefly, overlapping primers described by h. geng et al. (genbank jx ) and others [ , ] were used to obtain the viral sequence. accuscript high fidelity reverse transcriptase (agilent technologies, inc., santa clara, ca) was used for first-strand cdna synthesis in the presence of superase-in rnase inhibitor (ambion). pcr was performed using phusion polymerase (new england biolabs) with denaturation steps performed at °c. the ′ and ′ ends of hcov-nl /rptec/ were determined from vrna using a race (rapid amplification of cdna ends) kit (rlm race, ambion, austin, tx) following the manufacturer's instructions. sequences were analyzed using an applied biosystem dna analyzer by using bigdye terminator (v. . ) chemistry and the same primers used for amplifications. the genomic sequence for isolate nl /rptec/ / was combined with other representative nl genomic sequences [ ] available in genbank (ncbi.nlm.nih.gov/ genbank/index.html) to build the final dataset. full genome alignments were performed using mafft . [ ] followed by minor manual adjustments in clustalw [ ] . the e-ins-i alignment strategy was used with the following parameters: scoring matrix (blosum ), gap open penalty ( . ), and offset value ( ). the aligned dataset was imported into jmodeltest version . . [ ] and the akaike information criterion (aic) was used to select a best-fit model of evolution for phylogenetic analysis. phylogenetic trees were constructed using mrbayes . . [ ] . the markov chain was run for a maximum of million generations, with a stopping rule implemented so that the analysis would halt when the average deviation of the split frequencies was < . . four independent analyses were conducted, each with cold and heated chains with the default heating parameter (temperature = . ). every generations were sampled and the first % of mcmc samples discarded as burn-in. hcov-nl /amsterdam- hcov-nl /amsterdam- was obtained from the biodefense and emerging infections research resources repository (bei resources, manassas, va). plaque assays were performed following the procedures outlines in references and . the art of animal cell culture for virus isolation methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus bovine viral diarrhea disease associated with a contaminated vaccine demonstration and genotyping of pestivirus rna from mammalian cell lines bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines identification of pestiviruses contaminating cell lines and fetal calf sera bovine polyomavirus, a frequent contaminant of calf serum bovine polyomavirus, a frequent contaminant of calf sera a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species identification of novel porcine and bovine parvoviruses closely related to human parvovirus the association of calf serum with the contamination of bhk clone suspension cells by a parvovirus serologically related to the minute virus of mice (mvm) replication of bovine herpesvirus type in human cells in vitro bovine herpesvirus type : a special herpesvirus (review article) an adventitious viral contaminant in commercially supplied a cells: identification of infectious bovine rhinotracheitis virus and its impact on diagnosis of infection in clinical specimens growth characteristics of bovine herpesvirus (infectious bovine rhinotracheitis) in human diploid cell strain wi- infection studies on human cell lines with porcine circovirus type and porcine circovirus type investigations of porcine circovirus type (pcv ) in vaccine-related and other cell lines evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products torque teno sus virus (ttsuv) in cell cultures and trypsin presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle comparative analysis of the full-length genome sequence of a clinical isolate of human parainfluenza virus b mutations in the gm binding site of simian virus vp alter receptor usage and cell tropism atcc w ccl- ) have developed vacuoles. what is wrong? seroprevalence of cytomegalovirus infection in the united states detection of human cytomegalovirus dna, rna, and antibody in normal donor blood the histopathologic identification of cmv infected cells in biopsies of human renal allografts. an evaluation of transplant biopsies by in situ hybridization widespread presence of cytomegalovirus dna in tissues of healthy trauma victims understanding human coronavirus hcov-nl osterhaus ad: a previously undescribed coronavirus associated with respiratory disease in humans epidemiology and clinical presentations of human coronavirus nl infections in hong kong children the novel human coronaviruses nl and hku human coronavirus nl , a new respiratory virus identification of a new human coronavirus genomic analysis of colorado human nl coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene, and evidence of recombination human airway epithelial cell culture to identify new respiratory viruses: coronavirus nl as a model identification of human herpesvirus- as a causal agent for exanthem subitum human herpesvirus infection in hematopoietic stem cell transplant patients human cytomegalovirus herpes simplex virus and human cytomegalovirus replication in wi- cells. iii. cytochemical localization of lysosomal enzymes in infected cells generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium fatal hemorrhagic pneumonia concomitant with chlamydia pneumoniae and parainfluenza virus infection sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses a family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study novel human reovirus isolated from children with acute necrotizing encephalopathy. emerg infect dis morphogenesis of coronavirus hcov-nl in cell culture: a transmission electron microscopic study human herpesvirus- infection in children. a prospective study of complications and reactivation plaque assay for human coronavirus nl using human colon carcinoma cells systematic assembly of a full-length infectious clone of human coronavirus nl replication-dependent downregulation of cellular angiotensin-converting enzyme protein expression by human coronavirus nl extensive human cytomegalovirus (hcmv) genomic dna in the renal tubular epithelium early after renal transplantation: relationship with hcmv dnaemia and long-term graft function freeze-thawing of breast milk does not prevent cytomegalovirus transmission to a preterm infant identification of cell lines permissive for human coronavirus nl sars-associated coronavirus replication in cell lines inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme -a new cardiac regulator exogenous ace expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication preferential transformation of human neuronal cells by human adenoviruses and the origin of hek cells acute renal impairment in coronavirus-associated severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus multiple organ infection and the pathogenesis of sars tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases viral loads in clinical specimens and sars manifestations evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction isolation of a novel coronavirus from a man with pneumonia in saudi arabia is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku . emerg microb infect human herpesvirus infection in renal transplantation mafft version : improvement in accuracy of multiple sequence alignment clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice posada d: jmodeltest: phylogenetic model averaging mrbayes: bayesian inference of phylogenetic trees isolation and genetic characterization of human coronavirus nl in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells part of this work, including electron microscopy, was performed when the corresponding author was at the dept of pathology at the loyola university medical center (lumc), maywood, illinois. at lumc, linda fox provided excellent assistance with electron microscopy. some of this work was performed in partial fulfillment of internship requirements for em and mcl. the authors thank dr. gary heil for mdck-london cells. electron microscopy at the university of florida was performed by karen kelley. this work was financed by intramural funds made available to j. lednicky. the authors declare that they have no competing interests.authors' contributions jal conceived of the work, participated in all procedures, interpreted data; tbw performed phylogenetic analyses, interpreted data, and both jal and tbw wrote the manuscript, em, jcl, sbh, and mcl performed cell culture and virology work, and photographed cells; em assisted with dna and rna extractions, and ifa. jcl helped format the manuscript. all authors read and approved the final manuscript. key: cord- - ipzvryb authors: alagarasu, kalichamy; bachal, rupali v; bhagat, asha b; shah, paresh s; dayaraj, cecilia title: elevated levels of vitamin d and deficiency of mannose binding lectin in dengue hemorrhagic fever date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ipzvryb background: altered plasma concentrations of vitamin d and mannose binding lectin (mbl), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. the objective of the present study was to find out whether plasma concentrations of mbl and vitamin d are different in patients with dengue fever (df) and dengue hemorrhagic fever (dhf). the results: the plasma concentrations of vitamin d and mbl were assessed in df cases, dhf cases and apparently healthy controls using elisa based methods. vitamin d concentrations were found to be higher among both df and dhf cases as compared to healthy controls (p < . and p < . ). vitamin d concentrations were not different between df and dhf cases. when the dengue cases were classified into primary and secondary infections, secondary dhf cases had significantly higher concentrations of vitamin d as compared to secondary df cases (p < . ). mbl concentrations were not significantly different between healthy controls and dengue cases. mbl concentrations were observed to be lower in dhf cases as compared to df cases (p < . ). although mbl levels were not different df and dhf cases based on immune status, the percentage of primary dhf cases ( %) having mbl levels lower than ng/ml were less compared to primary df cases (p = . ). conclusions: the present study suggests that higher concentrations of vitamin d might be associated with secondary dhf while deficiency of mbl may be associated with primary dhf. dengue, caused by dengue virus (denv), constitutes a public health emergency of international concern. denv infection in humans results in a spectrum of outcomes ranging from asymptomatic to undifferentiated fever, mild form of the disease namely dengue fever (df) to severe forms including dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) that may be fatal [ ] . the outcome of denv infection is determined by multiple factors including viral virulence, host genetics and host immune responses [ ] . among the various components of host immune responses, t cells, antibodies, cytokine storm and complement factors contribute to the pathogenesis of dengue [ ] . epidemiological studies have shown an association between dhf/dss and secondary denv infection. preexisting antibodies and cross reactive t cell responses induced by the primary infection is believed to exacerbate the disease during secondary infection [ , ] . proinflammatory cytokines namely interleukin- (il- ), tumor necrosis factor-α and interferon-γ and anti inflammatory cytokine il- also contribute to dengue disease pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . activation of t cells, antibodies and cytokines are influenced by various immunomodulators. increase or decrease in the levels of these immunomodulators influences the outcome of viral infections [ ] . vitamin d is a potent immunomodulator affecting both innate and adaptive immune responses. vitamin d binds to vitamin d receptor (vdr), translocates to the nucleus and influences gene expression. vitamin d enhances the phagocytic capacity of macrophages and induces antimicrobial peptide gene expression contributing to innate immune responses [ ] . vitamin d inhibits t-helper (th ) cell and cytotoxic t cell responses. it decreases b-cell proliferation, plasma-cell differentiation and igg secretion [ ] . vitamin d also enhances th cytokine and il- responses [ , ] . vitamin d deficiency increases the risk of cancer, tuberculosis, as well as influenza and human immunodeficiency virus infection [ , , ] . vitamin d has been reported to influence the expression of denv receptors in immune cells [ ] [ ] [ ] . a study from vietnam has shown the association of vitamin d receptor gene polymorphisms with susceptibility to dhf [ ] . one of the major pathways of complement activation is initiated by binding of the virus to mannose binding lectin (mbl). mbl is a pattern recognition molecule that recognizes specific sugar molecules present on the surface of microorganisms including denv [ , ] . point mutations in the mbl gene lead to reduced concentrations of functional oligomers. genetically determined variation in serum concentrations of mbl has been shown to influence the susceptibility to infectious, autoimmune and cardiovascular diseases [ ] . alleles of mbl gene that are associated with higher concentrations of functional mbl, have been shown to be associated with thrombocytopenia in dengue infected patients [ ] . mbl concentrations were also found to be increased in acute samples of dhf cases as compared to df cases [ ] . since mbl and vitamin d are known to influence innate and adaptive immune responses and denv pathogenesis is immune mediated, we hypothesized that altered levels of plasma vitamin d and mbl might be associated with dengue disease severity. therefore, we investigated the levels of plasma vitamin d and mbl in dengue infected patients in the context of disease severity and immune status. among the patients included in the study, based on the df/dhf defining criteria of the world health organization (who) [ ] , had df and had dhf. males were over represented in both df and dhf patients. the male to female ratio in df was : . and in dhf, it was : . demographic and clinical characteristics of patients were given in table . the median age of dhf cases ( . years) was significantly lower than that of df cases ( . years) (p = . ). the number of primary cases with df ( . %) was higher than the number of primary cases with dhf ( . %) (p = . ). when the presence of clinical symptoms was compared between df and dhf cases, the presence of fever with chills, headache, myalgia, arthralgia, retro orbital pain and rash were reported equally in df and dhf cases. presence of nausea/vomiting and abdominal pain was represented by the dhf cases. thrombocytopenia was significantly over represented in dhf cases as compared to df cases (p < . ). the median count of platelets was significantly lower in dhf cases (p = . ) ( table ) . among the dhf cases, gastrointestinal bleeding, manifested by melena or hematemesis was reported in ( . %) cases, hematuria was observed in six ( . %), gum bleeding in seven ( . %), conjunctival hemorrhage in one ( . %) and epistaxis in one ( . %). plasma leakage was observed in ( %) patients either as ascites (n = ) and/or as pleural effusion (n = ). shock/hypotension was observed in five patients ( . %). no fatality was observed in the cases included in the study. plasma -hydroxy vitamin d (vitamin d) concentrations were investigated in df cases, dhf cases and healthy controls. vitamin d concentrations were found to be significantly higher in df and dhf cases as compared to healthy controls (healthy controls vs. df cases p < . ; healthy controls vs. dhf cases p < . ). among df and dhf cases, the vitamin d concentrations were found to be higher in dhf cases, though, the difference was not statistically significant (p > . ) ( figure ). the sample size has a power of . and . to detect the differences observed between dhf and healthy controls and between df and healthy controls respectively. when the patients were grouped based on immune status and disease severity, secondary dhf cases had significantly higher concentrations of vitamin d as compared to secondary df cases (p < . ). the sample size has a power of . to detect the differences observed between secondary df and secondary dhf cases. vitamin d concentrations were not significantly different between primary df and primary dhf cases (p > . ). when comparisons were made between primary df and secondary df or primary dhf and secondary dhf, vitamin d levels were not different (p > . ) ( figure ). plasma mbl concentrations were assessed in df cases, dhf cases and healthy controls. mbl concentrations were not significantly different between healthy controls and df or dhf cases (p > . ). when df and dhf cases were compared, significantly lower concentrations of mbl were observed in dhf cases (p < . ) (figure ). the sample size has a power of . to detect the observed differences between dhf and df cases. the sample size is underpowered to detect the differences (effect size of less than . ) observed between df and healthy controls or dhf and healthy controls. however, the sample size has a power of above . to detect an effect size of . and above. when the patients were classified into primary and secondary cases and compared, irrespective of df or dhf, mbl concentrations were not different (p > . ). mbl concentrations were lower in primary dhf cases as compared to primary df cases though not statistically significant. mbl levels were not different between secondary df and secondary dhf ( figure ). in the present study, plasma concentrations of vitamin d and mbl were investigated in dengue patients from pune, maharashtra, western india. pune is endemic to dengue with - cases occurring annually. all four serotypes have been reported to be circulating in pune [ ] . the demographic and clinical characteristics reported in the present study were similar to that reported in our earlier study [ ] . investigation of vitamin d concentrations revealed significantly higher concentrations of vitamin d in dengue patients (both df and dhf) as compared to apparently healthy controls suggesting that higher concentrations of vitamin d might be associated with symptomatic disease. further analysis revealed that the association was more evident in secondary dhf. it has been shown that vitamin d induces expression of dendritic cell specific intercellular adhesion molecule grabbing non integrin (dc-sign), the primary receptor for denv entry into immature dendritic cells [ ] . it has also been shown that calcitriol (active form of vitamin d) increases the expression of fcγ receptors on human monocytic cell lines and monocyte derived dendritic cells [ , ] . the increased concentrations of vitamin d, in denv infected cases with secondary infection, might enhance viral entry through increased expression of fcγ receptors leading to higher viral load, uncontrolled inflammatory responses and subsequent development of dhf. vitamin d is also known to suppress th cytokines and enhance il- production by peripheral blood mononuclear cells in response to microbial antigens [ ] [ ] [ ] . since il- is known to play a role in dengue disease pathogenesis [ ] , it is possible that vitamin d could also contribute to disease pathogenesis through altered il- response. since the effect of vitamin d is also dependent on single nucleotide polymorphisms in the vdr gene [ ] , the influence of vitamin d on dengue in the context of host genetics needs to be investigated. analysis of circulating concentrations of mbl in dengue cases and healthy controls revealed no significant difference between the two groups suggesting that the mbl mediated pathway of complement activation might be inhibited or may not be induced during denv infection. deficiency of mbl has been reported in patients infected with other rna viruses such as crimean-congo hemorrhagic fever virus, respiratory syncytial virus and severe acute respiratory syndrome (sars) corona virus [ ] [ ] [ ] . interaction between complement components and non structural protein (ns ) of flaviviruses has been reported to inhibit classical and lectin pathways of complement activation [ ] . the present study revealed significantly lower levels of mbl in dhf cases suggesting that reduced activation of mbl mediated complement pathway might be associated with dhf. mbl is known to interact with n-linked glycans of structural proteins of denv and neutralize the virus by blocking viral fusion. in experimentally infected mice, mbl dependent intravascular clearance of denv has also been reported [ ] . therefore, it is possible that mbl deficiency might have led to decreased activation of mbl mediated pathway of complement and reduced intravascular clearance of denv leading to higher viral load. higher viral load has been shown to be associated with dhf in several studies [ , ] . in the present study, deficiency of mbl was more evident in cases with primary dhf. the association of mbl deficiency with dhf in primary infection also suggests that the protective effects of mbl and the innate immune responses are more important during primary infection which could be overshadowed by presence of cross reactive complement fixing antibodies during secondary infection [ ] . in contrast to the present study, a study from brazil has reported higher concentrations of mbl among dhf cases [ ] . a higher mbl concentration might also lead to increased inflammation through enhancement of the production of pro-inflammatory cytokines. increased concentration of factor d and decreased concentration of factor h have been reported in dhf cases suggesting that imbalance in the regulation of factors h and d of the alternative pathway of complement activation is associated with dhf [ ] . since mbl concentrations are dependent on the presence of mutations in the structural and promoter regions of mbl gene, it is possible that variant alleles of mbl gene polymorphisms might be associated with dhf. a case control study from brazil has shown the association of wild type alleles of the mbl gene with thrombocytopenia in dengue patients [ ] . further case control studies are needed to confirm the phenotypic effects of mbl gene polymorphisms in dengue patients with primary infection from india. although, the present study is sufficiently powered to detect intermediate to large effect size, with regard to mbl, the study is underpowered and further studies with larger sample size are needed to confirm the preliminary associations. the present study suggests that higher concentrations of vitamin d are associated with secondary dhf. this association may be related to the inducing effect of vitamin d on fcγ receptor expression which might subsequently lead to higher viral load in dengue cases with secondary infection and hence development of dhf. the study also suggests that mbl deficiency is associated with primary dhf. this association might be related to the reduced activation of the mbl pathway of complement leading to higher viral load in dengue cases with primary infection. this is the first study that correlates the concentrations of vitamin d and mbl with immune status of dengue cases. blood samples from patients with dengue like illness were referred to the national institute of virology for diagnosis. samples were transported on ice and plasma was separated and aliquoted. one aliquot was used for dengue specific igm elisa and leftover aliquots were stored in - °c. a total number of samples, which were positive for dengue specific igm elisa, were included in the study. all the samples were collected during the seasonal outbreak in pune in . clinical presentations of the patients recorded by the clinicians were used to classify the patients. the patients were classified into those with df and those with dhf. patients with fever, headache, myalgia, retro-orbital pain, and rash were defined as df. dhf patients were categorized by the presence of at least two of the dhf defining criteria of the who [ ] : hemorrhagic tendencies/manifestations, thrombocytopenia, and evidence of plasma leakage. samples from apparently healthy blood donors were also used in the study. this study was approved by the national institute of virology human ethics committee. waiver of the informed consent was granted by the committee on the basis of "use of leftover specimens after clinical investigation" under the indian council of medical research guidelines . the in-house national institute of virology (niv) igm capture elisa kit was used to detect denv-specific igm. a known positive (p) and a known negative (n) serum control were used in every test. a sample showing p/n ratio > . times the optical density was considered positive. the igg capture elisa (e-den g, panbio, windsor, australia) was used to classify the cases into primary or secondary denv infection. igg levels of > units (defined by the manufacturers) indicated secondary infection. -hydroxyvitamin d (vitamin d) was quantitated in the plasma samples using an enzyme immunoassay kit (ids ltd, uk) according to the manufacturer's protocol. calibrators and controls were used in each assay. the percent binding (b/b%) of each calibrator, control and unknown samples were calculated by the following formula: b/b % = mean absorbance/mean absorbance for ' ' calibrator x . a calibration curve with b/b% and vitamin d concentrations were used to find out the concentrations of vitamin d in unknown samples in nm/l. the detection limit of the kit is nm/l. estimation of plasma mbl concentrations was done using the mbl oligomer elisa kit (bioporto diagnostics, denmark) according to the manufacturer's instructions. calibrators were used in each assay and a calibration curve was constructed by plotting the mean absorbance values for each calibrator on the y-axis against the corresponding mbl concentrations in ng/ml on the x-axis. the mbl concentration of each diluted plasma sample was then found by locating the point on the curve corresponding to the absorbance value of each diluted plasma sample and reading its corresponding concentration in ng/ml from the x-axis. the concentration of mbl in the undiluted plasma sample is calculated by multiplying this result by the sample dilution factor. the detection limit of the kit is ng/ml. using the statcalc program (epi info version . . , cdc, atlanta, ga, july ), the chi-square test with yates correction or fisher exact test (when any cell value was less than ) was performed to examine differences in demographic and clinical characteristics of the dengue patients. age and platelet counts were compared using mann-whitney u test. concentrations of mbl and [oh]d in plasma samples were compared between study groups using kruskal-wallis test with dunn's multiple comparison for selected groups. the p values from dunn's multiple comparison for selected groups were provided. since mbl levels varies depending on the presence of polymorphisms in the mbl gene, categorization of study subjects based on mbl deficiency (defined by a cutoff value of < ng/ml) was done and compared between study groups using the chi-square test with yates correction or fisher exact test. this cutoff value (< ng/ml) has been earlier shown to be a reliable predictor of low producing mbl genotypes using receiver operating characteristic analysis with individual data from healthy subjects from studies [ ] . all statistical analysis was performed using graphpad prism (version ). a two tailed p value less than . was considered significant. power calculations were performed using the software g*power version . . . the achieved power for significant results were calculated using the wilcoxon-mann-whitney test (two groups) option available in 't' test family of the software. the sample size, level of significance and effect size were provided as input. for calculating the effect size, the software uses mean values with standard deviation of the two groups [ ] . the authors declare that they have no competing interests govt of india: zoonosis division, national institute of communicable diseases dengue virus pathogenesis: an integrated view t lymphocyte response to heterologus secondary dengue virus infections antibodies determine virulence in 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advantage of: key: cord- -okh i l authors: wang, bin; yu, hai; yang, fu-ru; huang, meng; ma, ji-hong; tong, guang-zhi title: protective efficacy of a broadly cross-reactive swine influenza dna vaccine encoding m e, cytotoxic t lymphocyte epitope and consensus h hemagglutinin date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: okh i l background: pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. it is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. in this study, two forms of dna vaccines were constructed, one was made by fusing m e to consensus h ha (mha), which represents the majority of the ha sequences of h n swine influenza viruses. another was made by fusing m e and a conserved ctl epitope (np - ) to consensus h ha (mnha). their protective efficacies against homologous and heterologous challenges were tested. results: balb/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two dna vaccines. it was shown that the two vaccines elicited substantial antibody responses, and mnha induced more significant t cell-mediated immune response than mha did. then two h n strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). results indicated that both of the dna vaccines prevented homosubtypic virus infections completely. the vaccines’ heterologous protective efficacies were further tested by challenging with a h n swine influenza virus and a reassortant pandemic strain. it was found that mnha reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to mha and control groups. conclusions: the combined utility of the consensus ha and the conserved m e and ctl epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. this may provide a basis for the development of universal swine influenza vaccines. swine influenza virus (siv), member of genus influenza a virus of the family orthomyxoviridae, is a common and important causative pathogen involved in the porcine respiratory disease. mortality of siv-infected pigs is low, but morbidity may approach %. clinical signs of swine influenza include high fever, lethargy, anorexia, coughing, labored breathing and nasal discharge. synergistic or secondary infections with opportunistic organisms may increase the severity of clinical disease [ ] . beyond the veterinary implications, influenza virus infections in pigs also present an important public health risk. since pigs express sialic acid receptors for both mammalian and avian strains of influenza viruses on their tracheal epithelial cells [ ] , they could potentially serve as "mixing vessels" for the generation of new reassortant strains of influenza viruses that have pandemic capacity. the recently emerged pandemic h n which resulted in over , deaths is an example (http://www.who.int/csr/don/ _ _ /en/ index.html). genetic analyses revealed that this pandemic h n influenza virus is a triple reassortment of multiple strains of viruses circulating in the north america and eurasia swine population [ , ] . therefore, siv-infection control would be of benefit to both reduce the economic losses of swine industry and human health. vaccination is considered to be the most effective method to control sivs. currently, commercially available swine influenza vaccines are adjuvanted, wholevirus killed vaccines. although the vaccines reduce the severity of disease and the extent of virus shedding in pigs after challenge, they do not provide consistent protection from infection [ ] . the vaccines function by targeting the surface glycoprotein hemagglutinin (ha), the most variable influenza virus antigen. protective efficacy depends on the antigenic match degree between vaccine and circulating strains. however, influenza viruses continuously evolve by increasing the mutations in epitopes (antigenic drift) or by reconstituting the genome with other strains (antigenic shift). the increased incidence of avian-like or human-like siv reassortants, which exhibit great genetic diversity and thus antigenic diversity with classical sivs, has been documented [ ] [ ] [ ] , resulting in the "antigenic mismatch" between vaccine and the circulating strains. in addition, each of the dominant subtypes circulating in swine population worldwide, i.e. h n , h n and h n , has developed multiple genetic clusters based on the phylogenetic analysis of ha genes [ ] [ ] [ ] [ ] . previous studies have demonstrated that some viruses in different clusters showed only limited cross-reactivity [ , ] , suggesting that the genetic and antigenic heterogeneities within each subtype may reduce the vaccines' effectiveness [ ] [ ] [ ] [ ] . vaccines that induce broad protective immunity against diverse siv clusters and even subtypes, or limit the spread of the viruses between pigs and humans, especially for pandemic strains, are therefore needed. consensus has of different strains within one subtype were previously studied for eliciting cross-cluster protection [ ] , but ha induces predominately a subtype-specific humoral immune response. in contrast, the conserved viral antigens, such as m e and internal np, can generate heterosubtypic immunity protective against diverse virus strains and subtypes [ ] [ ] [ ] [ ] . unlike ha, the np-induced cell-mediated immune responses do not prevent infection, but reduce the severity of illness and accelerate the virus clearance. it is reasonable to propose that the combined utility of the consensus ha and conserved viral proteins will confer complete protection against homosubtypic and, at least partial, heterosubtypic challenge. in the present study, two types of swine influenza dna vaccines were constructed: ( ) fusing matrix ectodomain (m e) to consensus ha of h subtype siv (mha, means m e + ha) and, ( ) fusing m e and a conserved ctl epitope, np - , to consensus h ha (mnha, means m e + np - + ha). then their protective efficacy and broadness against divergent h n and h n siv challenges were tested in mice. the dna vaccines elicited m e-and ha-specific antibodies to increase the expression level, the m e, ctl epitope and consensus h ha gene was optimized for codon usage, rna structure and gc content. then the codonoptimized consensus ha, linked with m e, was cloned into eukaryotic expression vector with or without np - to generated two dna vaccines ( figure ). after nucleotides sequencing, the dna vaccine plasmids were transfected into human embryonic kidney (hek) t cells,the expression of each chimeric protein was confirmed by indirect immunofluorescence assay (ifa). ifa showed that both m e and ha genes expressed in vitro (data not shown). then the two dna vaccines, as well as empty vector, were coated with gold particles and delivered into the skin with a gene gun. humoral immunity was analyzed by detecting the presence of antigen-specific antibodies. as can be seen in figure , each of the constructs evoked a substantial ha-specific igg response after the booster injection, suggesting that the two vaccines were adequately delivered and expressed in mice. no significant difference in serum igg antibody levels were observed between mha and mnha group (p > . ), indicating that the addition of a -mer length ctl epitope did not influence the vaccine's antibody-inducing ability significantly. but the antibody titer of m e was lower than that of ha (p < . ). this is not surprising, because m e contains only amino acids and it was reported that m e is less immunogenic than ha [ ] . hi assays were then performed on both homosubtypic and heterosubtypic virus to assess the ability of inducing relevant and cross-reactive functional antibodies. as detailed in table , both of the vaccine immunization groups developed hi antibodies against homologous viruses, but induced very low levels of hi antibodies against heterologous viruses. the ability of the clt epitope-containing vaccine, mnha, to induce cellular immune response was determined by ifn-γ elispot assay. a synthesized peptide np - was used for restimulating splenocytes isolated from mice of each group. results showed that, after restimulation, the number of activated ifn-γ secreting cells from mice immunized with mnha was significantly higher than that from mice immunized with mha and empty vector (p < . , figure ). both mha and mnha vaccination conferred complete protection against homologous challenge, but mnha showed more significant cross-protection against heterologous challenge than mha did our previous study indicated that the wild type of swgd strain (h n ) replicates poorly in mice (unpublished data). to make the virus be suitable for vaccine efficacy assessment in vivo, we constructed a recombinant virus (r ) by using reverse genetics. r contains ha and na from swgd , and the internal proteins from pr . balb/c mice were infected intranasally with two homologous strains belonging to different clusters within h subtype days after the last immunization [ ] . mice lungs were taken days post infection for virus titration and histopathologic changes observation. results demonstrated that there was no detectable virus load in all the vaccine-immunized mice, while empty vector control group showed high lung viral titers ( figure ). in addition, histopathologic observation showed no obvious histopathologic changes in vaccinated mice. in contrast, the empty vector control group exhibited histopathological damages including the dropout of mucous epithelium cells, interstitial edema, hyperemia, hemorrhage, and inflammatory cell infiltration ( figure ). since m e and np - are conserved in different subtypes of influenza viruses, we hypothesized that the dna vaccine can confer partial protection against heterosubtypic challenge. to test this, the vaccines' protective ability was studied in mice challenged with h n sivs, which are also circulating in swine population worldwide. the immunized mice were challenged with heterologous g and rpan , respectively. lung viral titers determination and pathological analysis were performed as described above. results indicated that all the mice that had been vaccinated with mha had a detectable virus level, although showed a reduction in mean viral titers in both challenge groups compared with vector control, the reduction did not reach significance (p = . for rpan group and p = . for g group, figure ). histopathological analysis exhibited corresponding results, i.e., mice of mha group had lung pathology as almost severe as vector inoculated group. in mnha group, although the mice also exhibited detectable gly-gly-gly-gly-ser-gly-gly-gly-gly-ser-gly-gly-gly-gly-ser linker. virus shedding, the mean viral titers had been significantly reduced in both rpan -and g -challenged groups compared to vector-immunized control (p < . ), showing average viral titer reduction of . and . logs, respectively ( figure ). as expected, pathology damage of mice lung of mnha group was less pronounced than that of mha and vector control group, moderate histopathological lesions, mainly vascular congestion and various degrees of hemorrhage were observed ( figure ). collectively, these findings indicate consensus ha in combination with m e elicits cross-cluster immunity, and ctl response contributes to the control and clearance of infection and reduces pathogenesis. although the viruses are highly and continuously variable because of antigenic drift and shift, influenza is a vaccinepreventable disease. the conventional inactivated human influenza vaccines are updated annually with the world health organization-recommended h n , h n , and influenza b strains in order to antigenically match the viruses predicted to be the most likely to cause the next epidemic. unlike human influenza vaccines, strains used for swine influenza vaccines preparation are not regularly replaced, making the vaccines provide limited protection against antigenically diverse sivs. a universal vaccine designed based on the conserved viral proteins that confers broad protection is an attractive solution to counter the features of highly variable of not only human but also swine influenza viruses. many studies developed universal vaccines against human influenza, whereas broadly responsive swine influenza vaccines, to our knowledge, have not been reported. the virus envelope glycoprotein ha is the most abundant surface protein, antibodies against ha can provide protection by blocking virus attachment and entry. studies from several groups have demonstrated the antiviral efficacy of consensus ha as an "intra-subtype" universal vaccine. the study of chen et al. [ ] showed that the consensus h ha-based dna vaccine elicited only moderate neutralization activities toward the h n clade . and clade . viruses, and provided not complete, albeit significant, protection against clade . virus, suggesting that consensus ha alone may be not enough to induce complete protection against all strains within one subtype, much less heterosubtypic strains. however, ideally, universal vaccines should be capable of inducing protection against both homosubtypic and heterosubtypic challenges. to this end, we linked m e and ctl-inducing epitope np - to consensus h ha to construct a universal swine influenza vaccine. the reasons we chose m e and np - are: ( ) both of them are highly conserved among different siv strains, np - are even conserved among human, avian and swine figure protection of mice from homologous and heterologous challenges. mice lung viral titers at day after challenges were determined in eggs from an initial dilution of : in phosphate-buffered saline and expressed as eid /ml. the limit of virus detection was . log. influenza strains (table ) , they were added in view of their ability to broaden the protective scope of the vaccine. ( ) np - induces a vigorous ctl response [ ] . studies indicated that specific ctls provide some level of crossprotection against antigenically distinct viruses of not only the same subtype but also different subtypes [ , ] . previously we have shown that swhlj and swgd had evolved into independent lineages based on the phylogenetic analysis of the ha gene. to see whether the two strains were antigenically distinct, cross-hi assay was performed by using r -specific anti-serum. it was found that the titer of cross-reacting hi antibodies (swhlj -reacting) was significantly lower than that of r -reacting hi antibodies (p < . , data not shown), thus suggesting that they belong to different antigenic clusters. results of present study demontrated that both mha and mnha vaccination provided complete protection against these two viruses challenges, cross-cluster protections were therefore suggested. while mnha, but not mha, induces immune responses partially protect against heterologous influenza infection, suggesting the echanced t-cell mediated response is not required for homologous protection. results also further confirm that ctl responses play important roles for heterologous protection, meaning that strengthening ctl responses are promising ways for universal influenza vaccines development. it was established that the pandemic h n virus is pathogenic and is readily transmitted in pigs [ ] [ ] [ ] [ ] . although the new h n virus is now considered to be post-pandemic, the possibility that it recombines with other influenza viruses in pigs then yielding a novel potential epidemic or pandemic strain still exists [ ] . here in this study, the mice were partially protected from the reassortant pandemic virus challenge after the immunization of mnha. the vaccine immunization inhibited the pulmonary viral replication significantly in mice, thus could accelerate the virus clearance, and reduce the potential of transmission and the risk of recombination. although the heterologous protection elicited by the dna vaccine was not complete, this methodology has a large potential for improvements. the present study lays the foundation for universal swine influenza vaccine development, and call for further investigations in which the heterologous immune response should be further enhanced, such as the addition of molecular adjuvants [ , ] and/or more copies of conserved viral protein encoding genes [ ] , and the usage of dna-prime protein/virus-boost immunization schedule [ , ] . a previous study conducted by heinen et al. constructed a dna vaccine containing m e and full length of np genes, but ha encoding gene was not included. their data showed that the m e-np dna vaccination produced enhancement of disease after challenge [ ] . although the mechanism underlying which is unclear, we think the ha may play roles in this discrepancy between the results of heinen et al.'s and ours. another possible reason should be taken into consideration is that gene gun immunization induces more strong immune response that the conventional needle injection method, which was used in heinen et al.'s study [ ] . it is necessary to study whether the dna vaccines we developed here will confer protection in pig models. also, it would be interesting to study how, if does, the ha protein influence the vaccines' efficacy. the combined utility of the consensus ha and the conserved m e and ctl epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse models. this may provide a promising strategy for universal swine influenza vaccine development. viruses, cell and mice medicine, south china agricultural university. r , containing swgd surface glycoproteins and pr internal proteins, was prepared by using reverse genetics as described [ ] . these viruses were propagated in allantoic cavities of to -day old spf embryonated chicken eggs and stored at − °c until use. fifty-percent embryo infectious dose (eid ) titers were determined by serial titration in embryos and calculated by the method of reed and muench [ ] . hek t cells were maintained in dulbecco's modified eagle's medium (dmem, sigma) supplemented with % fetal bovine serum in humidified % co atmosphere at °c. spf female balb/c mice ( - weeks of age) were purchased from shanghai slac laboratory animal co., ltd., and maintained with free access to sterile food and water. all animal studies were conducted in accordance with the ethical guidelines and were approved by the ethical committee of shanghai veterinary research institute, chinese academy of agricultural sciences. a total of ha and m sequences with full-length of h n subtype siv were, respectively, downloaded from genbank database and aligned by megalign program supplemented in dnastar package (dnastar madison, wi). the consensus sequences were created based on the most common amino acid in each position of the alignment. after the consensus m sequence was generated, the first amino acids were selected as m e motif [ ] . then codons of the consensus ha, as well as m e and an immunodominant cytotoxic t lymphocyte (ctl) epitope of the np protein, np - (tyqrtralv) [ ] , were optimized for mammalian expression and synthesized by genscript corporation (nanjing, china). the (g s) linked chimeric orf encoding m e, ctl epitope and consensus ha was pcr-amplified by introducing ' sma i and ' nhe i restriction sites for ligation into the pcaggs vector, under the control of the cytomegalovirus (cmv) enhancer and chicken β-actin promoter (designated as mnha). similarly, mha was designed and constructed, except that the ctl epitope was omitted from the nterminal end of the consensus ha ( figure ). after the recombinant plasmids being identified by nucleotide sequencing, they were propagated in e. coli bacteria and purified using mega purification kit (qiagen, valencia, ca) for in vitro transfection as well as in vivo animal immunization. the final dna preparations were resuspended in nuclease-free water and stored at − °c until further use. hek t cells were seeded in -well plates and transfected at - % confluence with μg of mha, mnha or empty vector using lipofectamine transfection reagent (invitrogen), as recommended by the manufacturer. after h, the transfected cells were scraped from the culturing plate, washed with pbs, then spotted onto a glass slide, air dried, and fixed with pre-chilled acetone. upon removal of the residual solvents from the slides, the cells were incubated with anti-m e and -ha polyclonal antibodies for h at °c. a secondary alexa fluor conjugated goat anti-mouse igg antibody was then used to detect the primary antibody. fluorescence images were scanned using an inverted microscope after the samples were mounted by glycerol. gene gun immunization was performed as previously described [ , ] . mice received two nonoverlapping abdominal deliveries of antigen encoding plasmid-or empty vector-coated gold beads ( μm) at the shaved skin with a -wk interval. with each shot, μg of dna immobilized onto . mg gold particles was delivered at a helium discharge pressure of - psi with a helios gene gun (bio-rad). each test group contained mice with experiments organized as follows: ( ) mice of each group were used to complete ifn-γ elispot assays on day post the nd immunization; ( ) the left mice of each group were divided as subgroups, each comprised of mice, then intranasally challenged with eid of two h n (swhlj and r ) and two h n (g and rpan ) strains, respectively, at days after the final immunization. serum samples were collected from orbital bleeds on day post last immunization. m e and ha antibody titers were measured using indirect elisa. elisa plates were coated overnight with synthetic m e peptides or inactivated swhlj in . m carbonate buffer (ph . ) at °c and blocked with % non-fat milk for h at °c. a series of two-fold dilution of sera (starting dilution : for ha and : for m e, μl/well) were incubated at °c for h, followed by three washes with pbst. then hrp-conjugated goat anti-mouse igg (zhongshan biotechnology co., ltd beijing, china) and , '- , '-tetramethyl benzidine (tmb) was used for m -and ha-specific antibody detection and color development, respectively. the resulting optical density (od) at nm was determined with a plate reader after the reaction was stopped with m h so . hi assays were performed using . % chicken red blood cells (rbcs) with ha units of homologous (swhlj and r ) and heterologous (g and rpan ) virus and receptor-destroying enzyme (rde, sigma) treated serum, as previously described [ ] . the reciprocal of highest dilution of serum that gave complete inhibition of hemagglutination was considered the hi titer. each assay was performed in triplicate. a titer of less than (starting serum dilution) was assigned for serum samples that did not inhibit hemagglutination. the elispot assays were performed using mouse ifnγ elispot kits following methods recommended by the manufacturer (dakewe biotech, pr china). briefly, singlecell suspensions of freshly isolated spleen lymphocytes were seeded into the plates ( /well) pre-coated with anti-ifn-γ monoclonal antibody. cells were stimulated with synthesized np - peptide at a final concentration of μg/ml in a °c humidified incubator with % co . phytohemagglutinin (pha, μg/ml) and medium alone were used as positive and negative controls, respectively. after a h culture, plates were washed and incubated for h with biotinylated anti-mouse ifn-γ antibody. streptavidin-horseradish peroxidase was then added, ifn-γ spots were developed with -amino- -ethylcarbazole (aec) and counted using an automated elispot reader. results were expressed as spot-forming cells (sfc) per million cells. on day post-infection, mice from each group were sacrificed to collect lungs for virus titration and pathologic examination to determine the protective ability of dna vaccines. for virus titration, the whole lungs were homogenized in ml of sterile phosphate-buffered saline (pbs) containing . mg/ml of streptomycin and iu/ml of penicillin. the homogenates were diluted -fold serially after being centrifuged, then inoculated into embryonated chicken eggs. infection within individual eggs was confirmed using a standard hemagglutination assay, the viral titers were determined by the method of reed and muench. lung tissue histopathologic sections were made as described elsewhere [ ] . briefly, the removed lungs were fixed in % neutral buffered formalin, dehydrated and embedded in paraffin. five micrometer sections were cut and stained with hematoxylin and eosin (h&e), and reviewed for histopathologic changes. bad a / ), and the chinese research fund for non-profit research institutions dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study molecular basis for the 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to acknowledge drs. wen-bao qi and ming liao (college of veterinary medicine, south china agricultural university) for providing rpan virus. we also gratefully acknowledge dr. yan-jun zhou (shanghai veterinary research institute) for providing laboratory reagents. this study was supported by grants from china international sci & tech cooperation program ( dfb ), shanghai basic research program ( jc ), national scientific supporting program of china data of experimental and control groups were presented as the averages ± standard error (se) and evaluated by anova method, where statistically significant results were defined as having a p value of less than . . the authors declare that they have no competing interests. key: cord- -usl z ep authors: zheng, wen-zhi; wei, tian-li; ma, fen-lian; yuan, wu-mei; zhang, qian; zhang, ya-xin; cui, hong; zheng, li-shu title: human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in beijing, china date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: usl z ep background: hpyv is a novel human polyomavirus (hpyv), and neither its natural history nor its prevalence in human disease is well known. therefore, the epidemiology and phylogenetic status of hpyv must be systematically characterized. methods: the vp gene of hpyv was detected with an established taqman real-time pcr from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. the hpyv -positive specimens were screened for other common respiratory viruses with real-time pcr assays. results: the prevalence of hpyv was . % ( / ), and children ≤ years of age accounted for % ( / ) of cases. all hpyv -positive patients were coinfected with other respiratory viruses, of which influenza virus a (ifva) ( / , . %) and respiratory syncytial virus ( / , . %) were most common. all hpyv -positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from . to . copies/μl nasopharyngeal aspirate specimen. the most common symptoms were cough ( %) and fever ( . %). the complete -bp genome (bj strain, genbank accession number km ) was amplified and showed % identity to hpyv strain a. conclusions: the prevalence of hpyv was . % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time pcr. because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between hpyv and respiratory diseases. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the family polyomaviridae contains viruses that are among the smallest known to infect humans, in terms of both their particle sizes and genome lengths. the - nm nonenveloped icosahedral particles carry a circular doublestranded dna genome of approximately bp, which is divided into three functional regions: the noncoding control region, the early gene region encoding the large t antigen and the small t antigen, and the late coding region, encoding capsid proteins vp , vp , and vp [ ] . human polyomaviruses (hpyvs) have not been associated with any severe acute disease in healthy humans. however, the viral proteins expressed by polyomaviruses (pyvs) can initiate the transformation and immortalization of cultured cells and cause cancer in experimental animals [ ] . the hpyv family currently consists of members, including jcpyv [ ] , bkpyv [ ] , wupyv [ ] , kipyv [ ] , mcpyv [ ] , hpyv [ ] , hpyv [ ] , tspyv [ ] , hpyv [ ] , hpyv [ ] [ ] [ ] , stlpyv [ ] , hpyv [ ] and njpyv- [ ] . previous studies have indicated that a number of hpyvs are associated with human diseases, such as progressive multifocal leukoencephalopathy (jcpyv), hemorrhagic cystitis (bkpyv), merkel cell carcinoma (mcpyv), and trichodysplasia spinulosa (tspyv) [ , , , [ ] [ ] [ ] . a serological study demonstrated that the seroprevalence of these viruses in plasma samples from healthy adult blood donors was bkpyv ( %), jcpyv ( %), kipyv ( %), wupyv ( %), mcpyv isolate ( %); and mcpyv isolate ( %). the seroprevalence of all polyomaviruses in children under years of age (n = ) was similar to that in the adult population, suggesting that primary exposure to these viruses occurs in early childhood and seems to result in lifelong persistence [ ] . nevertheless, the natural histories of most hpyvs and their prevalence in human diseases are not yet well known. in , mcpyv was discovered by feng et al. [ ] . hpyv and hpyv were discovered with mcpyv in skin swabs from the foreheads of healthy volunteers [ ] . however, later research could not demonstrate a relationship between hpyv and merkel cell carcinoma or other skin diseases. a phylogenetic analysis of the complete hpyv genome indicated that hpyv shared a branch with kipyv and wupyv. previous reports have shown that genomic fragments of kipyv and wupyv have been regularly detected in nasopharyngeal aspirates of children with respiratory tract infections (rtis) and are suspected of a causal relationship with respiratory disease. however, the link between these pyvs and respiratory diseases remains speculative [ , , , ] . a real-time pcr assay was established here to determine the prevalence of hpyv throughout a time period of months (from october to september ). the prevalence of hpyv was . % ( / ). a complete hpyv genome was amplified, sequenced and found to be identical with a hpyv isolate from usa. with the standard curve derived from serial dna dilutions, the dynamic range of the real-time pcr assay was - copies/μl and the limit of detection was one copy. the coefficient of determination (r = . ) showed a good linear correlation. the taqman-based real-time pcr assay to detect hpyv did not amplify any other viral pathogen, showing the excellent specificity of this assay. four different dna concentrations ( - copies per reaction) were repeated five times in each run. the maximum coefficient of variation was . %, which indicates good precision (data not shown). a total of npa samples were obtained from children with rti. the sex ratio (male:female) was : ( . : ) and the median age was months (ranging from days to years). the prevalence of hpyv was . % ( / ) in the npa specimens tested. of the hpyv -positive patients, eight were male ( / , . %) and seven were female ( / , . %), so the prevalence was similar in both sexes (p > . ). the ages of the infected patients ranged from days to years, and children ≤ years of age accounted for % ( / ) of the total hpyv -positive children. the age distribution of the hpyv -infected children indicated that those aged - months had the highest infection rate table ) . all hpyv -positive patients were diagnosed with lower rti, including bronchopneumonia in nine ( %), acute bronchitis in three ( %), bronchitis in two ( . %), and pneumonia in one ( . %). the most common symptom was cough, which occurred in all patients ( %). other clinical presentations included fever (n = , . %), gasping (n = , %), vomiting (n = , %), and diarrhea (n = , . %). among the hpyv -positive specimens, the hpyv genome copies ranged from . to . copies/μl npa on a realtime pcr assay ( table ). the hpyv genome copy number was . copies/μl npa in children suffering lower rtis only, and was slightly higher than . copies/μl npa in those infected with lower rtis and other diseases, but these did not differ significantly (p > . , mann-whitney u test). to determine the complete hpyv genomic sequence, overlapping genomic fragments were amplified with nested pcr using pairs of virus-specific primers (listed in additional file : table s ), and the complete -bp genome was compiled with bioedit . software. the genome sequence of hpyv was deposited in genbank under accession number km . blast analysis with the complete hpyv genome showed a high level of nucleic acid identity to the six full hpyv genomes available in genbank. relationsship to the hpyv strain a was %. hpyv , thought to be a skin-tropic polyomavirus, was initially described in . since then, subgenomic fragments of hpyv dna have been detected in a variety of specimen types, including skin, respiratory secretion samples, and various tumor samples (for instance, the answers to these questions will require more information on the biology and epidemiology of the hpyvs. the genomes and proteins of hpyv , one of the novel hpyvs, show little sequence homology with previously reported hpyvs (bkpyv and jcpyv). although hpyv encodes a conserved, potentially carcinogenic ltag, previous studies have shown no association between hpyv and tumors [ ] . furthermore, a phylogenetic analysis indicated that ltag of hpyv (km ) is only distantly related to its homologues in other cancerassociated hpyvs. the hpyv , wupyv, and kipyv strains formed a clade in the complete genome and vp amino acid phylogenies, whether hpyv also associate with respiratory infection which need more clinical and experimental evidences to support. hpyv has been detected in specimens from the human respiratory tract, but there are as yet insufficient epidemiological data to demonstrate a correlation between hpyv and respiratory disease. because initial infections with most hpyvs occur in infancy, the prevalence of hpyv in npas from children was detected with real-time pcr. hpyv displayed an overall prevalence of . % in npa samples collected from children in a hospital in china, which is similar to its prevalence reported previously ( . - %) [ , ] . it has not been confirmed that hpyv infects humans via the respiratory tract, but the respiratory tract may be a possible route of transmission. in this study, hpyv was mainly detected in children less than years of age, and the peak incidence occurred in spring. all hpyv -positive patients were coinfected with other respiratory viruses, of which ifva and rsv were the most common. the hpyv -positive patients were diagnosed with lower rtis, % had bronchopneumonia, and the most common symptoms were cough and fever. although known hpyvs cause disease in patients with immune-system imbalances, they do not seem to cause obvious illnesses in the great majority of infected individuals. however, bkpyv can induce nephropathy in kidney transplantation recipients and jcpyv causes progressive multifocal leukoencephalopathy. progressively increasing or high viral loads are also associated with high-level viral replication and disease. for instance, progressive multifocal leukoencephalopathy and hemorrhagic cystitis are related to high viral loads of jcpyv and bkpyv, respectively. the present study cannot confirm that hpyv is the cause of rtis in hospitalized children, because the viral loads of hpyv were low ( . - . copies/μl) and the coinfection rate with other respiratory viruses was high. in previous reports, hpyvs were detected in the respiratory tract and skin but, to date, there has been insufficient evidence that hpyv is associated with any respiratory tract disease or skin disease [ ] . whether hpyv induce any disease requires the analysis of further data. the detection rate for hpyv by real-time pcr assay was . % in npa samples collected from hospitalized children with rti. an association between hpyv and respiratory diseases could not been revealed due to the high coinfection rate and the low hpyv viral load. from october, , to september, , nasopharyngeal aspirate (npa) specimens were collected continuously from hospitalized children with rti, who ranged in age from days to years, at beijing friendship hospital, beijing, china. the patients' parents or guardians gave their written informed consent for specimen collection and testing, and the project was approved by the ethical committee of the beijing friendship hospital. the npa specimens were collected and transported immediately to the national institute for viral disease control and prevention, china cdc, and stored at − °c until further processing. all demographic data and clinical findings were recorded on a standard form. total nucleic acids were extracted from each npa specimen using the qiaamp minelute virus spin kit (qiagen, beijing, china). a taqman-based real-time pcr assay for the detection of vp gene of hpyv was designed with primer express . software. the primer sequences used were hpyv -f '-ttaacacccttctttgtgctgcta- ' and hpyv -r '-gcccaattattcaaagcagctaa- ' , and the probe sequence was hpyv -p fam-ctgtcacag gcctgctgagcaatagatttc-tamra. the specificities of the primers and probe were evaluated in genbank with blast. the primers and probe were synthesized by invitrogen (beijing, china). a common reaction mix was prepared for the real-time pcr assays. briefly, the final μl reaction mix contained μl of taqman gene expression master mix, . μl of each primer ( pmol/μl), . μl of probe ( pmol/μl), μl of pmd -t/vp plasmid template (plasmid pmd -t linked to the vp gene of hpyv ), and . μl of h o. the amplification conditions included an initial incubation at °c for min and °c for min, followed by cycles of °c for s and °c for min, using the mx p qpcr system (agilent stratagene). ten-fold serial dilutions of the pmd -t/vp plasmid (from to copies/μl) were added to the real-time pcr reactions in duplicate. the results were used to generate a standard curve for hpyv . specificity was assessed by testing mixed samples of other common hpyvs, including wupyv, kipyv, jcpyv, bkpyv, and hpyv . to test the reproducibility of the assay, we added - copies/μl of pmd -t/vp plasmid to each reaction and each concentration of dna was repeated five times. in addition, housekeeping gene glyceraldehyd- -phosphate dehydrogenase (gapdh) was used as internal control. μl of nucleic acid of each npa specimen was added to each reaction. only samples that were positive according to both pcr and dna sequencing were considered "positive". fifteen overlapping fragments of the complete genome of hpyv strain bj were pcr amplified (table ) with the takara ex taq kit. the overlapping fragments were then cloned into pmd -t and sequenced (invitrogen). the nucleotide sequence of the full-length hpyv genome was then compiled using bioedit . software. the full-length hpyv sequence was aligned with the sequences of other hpyvs and other hpyv strains available in genbank with dnastar software. a neighbor-joining tree was constructed with mega . . the significance of differences between the prevalence rates and viral loads of various groups was tested with fisher's exact test and the mann-whitney u test. all analyses were performed with spss . software. genome analysis of the new human polyomaviruses human polyomaviruses in disease and cancer cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy new human papovavirus (b.k.) isolated from urine after renal transplantation identification of a novel polyomavirus from patients with acute respiratory tract infections identification of a third human polyomavirus clonal integration of a polyomavirus in human merkel cell carcinoma merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient a novel human polyomavirus closely related to the african green monkeyderived lymphotropic polyomavirus complete genome sequence of a tenth human polyomavirus discovery of a novel polyomavirus in acute diarrheal samples from children identification of mw polyomavirus, a novel polyomavirus in human stool discovery of stl polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique t antigen by alternative splicing identification of a novel human polyomavirus in organs of the gastrointestinal tract identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy association between a high bk virus load in urine samples of patients with graftversus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation progressive multifocal leukoencephalopathy and promyelocytic leukemia nuclear bodies: a review of clinical, neuropathological, and virological aspects of jc virus-induced demyelinating disease sv and human cancer: a review of recent data seroepidemiology of human polyomaviruses presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection molecular epidemiology of ki and wu polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus:assessment of assay sensitivity and specificity of real-time screening and typing methods wu and ki polyomaviruses in respiratory samples from allogeneic hematopoietic cell transplant recipients development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses human bocavirus in patients with respiratory tract infection pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement impact of human coronavirus infections in otherwise healthy children who attended an emergency department exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer prevalence of human polyomaviruses in common and rare types of non-merkel cell carcinoma skin cancer detection of novel polyomaviruses, tspyv, hpyv , hpyv , hpyv and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid human polyomaviruses in children undergoing transplantation no evidence for association of hpyv or hpyv with different skin cancers submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we thank the beijing friendship hospital, capital medical university, for providing the samples, and the laboratory staff who contributed to this study. no compensation was given to subjects for participation in this study. additional file : table s . primers and probes used to detect respiratory viruses. table s . primers used to amplify hpyv with nested pcr (docx kb) the authors declare that they have no competing interests. key: cord- - p x lwx authors: melnik, lilia i; garry, robert f; morris, cindy a title: peptide inhibition of human cytomegalovirus infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: p x lwx background: human cytomegalovirus (hcmv) is the most prevalent congenital viral infection in the united states and europe causing significant morbidity and mortality to both mother and child. hcmv is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (hiv)- infected patients with aids, and solid organ and allogeneic stem cell transplantation recipients. current treatments for hcmv-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit hcmv infection. the aim of this study was to develop therapeutic peptides targeting glycoprotein b (gb), a major glycoprotein of hcmv that is highly conserved across the herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing hcmv entry and infection. results: using the wimley-white interfacial hydrophobicity scale (wwihs), several regions within gb were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. the ability of synthetic peptides analogous to wwihs-positive sequences of hcmv gb to inhibit viral infectivity was evaluated. human foreskin fibroblasts (hff) were infected with the towne-gfp strain of hcmv ( . moi), preincubated with peptides at a range of concentrations ( nm to μm), and gfp-positive cells were visualized hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. peptides that inhibited hcmv infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. peptide - showed % inhibition of viral infection at a concentration of μm, and % and % inhibition at concentrations of μm and . μm, respectively. peptide - inhibited infection by % and % at concentrations of μm and μm, respectively, and % at a concentration of . μm. while peptides - and - , individually failed to inhibit viral infection, when combined, they showed % inhibition of hcmv infection at a concentration of . μm each. conclusions: peptides designed to target putative fusogenic domains of gb provide a basis for the development of novel therapeutics that prevent hcmv infection. human cytomegalovirus (hcmv) is a ubiquitous opportunistic pathogen that belongs to the betaherpesviridae. the virulence of this pathogen is directly linked to the immune status of its host. primary hcmv infection is generally asymptomatic in immunocompetent individuals, although it causes a mononucleosis-like syndrome in some. after primary hcmv infection, the virus establishes lifelong latency and periodically reactivates with notable pathological consequences. in contrast, hcmv infection in immunocompromised patients such as aids patients and solid organ and allogeneic stem cell transplantation recipients causes serious disease [ ] . primary infection of women during or right before pregnancy with hcmv is the most common cause of congenital viral infection leading to significant morbidity and mortality. congenital hcmv infection is also associated with spontaneous abortion, premature delivery, intrauterine growth restriction (iugr), and pre-eclampsia. the risk of primary infection in a seronegative mother is to %, which carries a to % risk of congenital infection [ , ] . the majority of congenitally infected babies are asymptomatic at birth; however, to % subsequently develop hearing defects or neurodevelopmental sequelae [ ] . although the most serious clinical sequelae are seen in cases where a mother acquires a primary infection during pregnancy, downstream side effects are also seen in cases where latent hcmv is reactivated [ ] and where a mother is reinfected with a different strain of the virus [ ] . hcmv has a double-stranded dna genome of kb encoding approximately genes [ ] . it has a very broad cellular tropism resulting in potential infection of nearly every organ system. the ability of hcmv to enter a wide range of cell types involves a complex interaction between several viral envelope glycoproteins and host cell surface receptors, although the entry of herpesviruses into host cells is still poorly understood. the hcmv virion envelope contains at least virusencoded glycoproteins that are involved in cell attachment and penetration [ ] . of these, glycoprotein b (gb) is the most abundant glycoprotein [ ] and is highly conserved among the herpesviridae [ ] . glycoprotein b plays a critical role in the hcmv entry process. initially, gb along with gm/gn, is involved in tethering of virions to heparan sulfate proteoglycans (hspg) on the surface of host cells. the short interaction of hcmv with hspg is followed by more stable interactions with one or more viral cellular receptors, namely epidermal growth factor receptor (egfr) [ ] , platelet-derived growth factor receptor (pdgfr) [ ] , and toll-like receptor tlr- [ ] . glycoprotein b also interacts with integrin αvβ , a coreceptor that enhances hcmv entry [ ] . integrins are known to synergise with egfr as well as with other receptors to activate signal transduction pathways [ ] [ ] [ ] . to complete the entry process, both viral and cellular membranes fuse, allowing the release of virion-associated tegument and capsid proteins into the cytoplasm. this final step of viral entry into host cells requires gb and the gh/gl complex [ ] [ ] [ ] [ ] . antibodies to hcmv gb have been shown not only to block penetration of virions into cells, but also to limit cell-to-cell infection, implying that gb plays a role in virion penetration into cells, cell-to-cell transmission, as well as fusion of infected cells [ , ] . recently, isaacson and coworkers used genetic complementation to confirm that gb is required for the fusion of viral and cellular membranes, virus entry, and cell-to-cell spread of hcmv [ ] . the importance of gb for viral infection suggests that this viral envelope protein may be a rational target for novel drug design. hcmv infection is highly prevalent in the population due to the ability of the virus to efficiently transmit between hosts that harbour and periodically shed the virus. hcmv is transmitted through direct exposure to infected bodily secretions, including saliva, urine and breast milk. following infection, hcmv enters the bloodstream and spreads to various organs including kidney, liver, spleen, heart, brain, retina, esophagus, inner ear, lungs, colon, and salivary glands [ ] . the ability of hcmv to infect a wide variety of cell types is not due to the presence of high plasma levels of extracellular virus, but is primarily due to cell-to-cell transmission between mononuclear phagocytes (possibly macrophages or dendritic cell precursors) and uninfected tissues [ ] . the lack of a successful hcmv vaccine as well as the toxicity and drug-induced resistance associated with current therapeutics for hcmv indicate that this virus continues to pose a significant public health problem. current treatments for hcmv disease target viral replication and can fail due to the emergence of drug-resistant virus variants and induction of adverse effects. hence, a new approach in drug design against hcmv is required [ , ] . since hcmv and other herpesviruses establish a lifelong latency in humans, antiviral therapy that inhibits viral entry may serve as an alternative to the already existing and inadequate therapeutic agents. here, we report the design, development and characterization of peptides that specifically inhibit viral infection and/or entry as a novel approach to prevent hcmv infection. structural studies place herpes simplex virus type gb- [ ] and epstein-barr virus gb into class iii viral fusion proteins (vfp) [ ] , which also includes vsv g [ ] , members of the gp superfamily (baculovirus and thogotovirus) [ ] and tentatively bornavirus g [ ] . because gb is the most highly conserved envelope protein amongst the mammalian and avian herpesvirues [ , ] , gb of hcmv is likely to be a class iii vfp and shares structural features with gb of other members of the herpesviridae. class iii viral fusion proteins share certain characteristics found in class i or class ii viral fusion proteins. the class iii viral fusion proteins contain an extended α-helix that trimerizes in the postfusion forms of the proteins [ , , ] , as has been well-documented for the post-fusion forms of the class i viral fusion proteins of orthomyxoviruses, retroviruses, paramyxoviruses, arenaviruses, and coronaviruses [ ] . similarly, the class ii viral fusion proteins of flaviviruses and alphaviruses contain a fusion domain comprised principally of β-sheets and "fusion loops." class iii viral fusion proteins also possess a fusion domain, as well as several other features of class ii viral fusion proteins, suggesting that these two classes of proteins may share a common progenitor [ ] . the class iii domain nomenclature used here can apply to both class ii and class iii viral fusion proteins: domain i (green), domain ii (yellow), domain iii (blue), domain iv (stem domain, indigo) ( figure ). this unified nomenclature assigns domain ii appellation to the following: vsv g domain iv in the nomenclature of roche et al. [ ] , hsv- gb- and baculovirus gp domain i in the nomenclature of heldwein et al. [ ] and kadlec et al. [ ] as the class iii fusion domain, which is structurally similar to class ii viral fusion proteins. in addition to minor adjustments in the ends of domains, the current class iii viral fusion protein numbering also combines two interacting domains into domain iii (i + ii in roche's vsv g nomenclature, iii + iv in heldwein's hsv- gb- nomenclature and kadlec's baculovirus nomenclature). the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally determined hydrophobicity scale that provides a quantitative description of a protein partitioning and folding into membrane interfaces. wwihs score-positive sequences may also interact with hydrophobic surfaces within proteins, and are often sequestered within pre-fusion forms of viral fusion proteins. in addition to similarities in the overall structure of the post-fusion forms of class iii vfp, there are additional similarities in the distribution of wwihspositive sequences (figure , red). the similarities include at least one extended "fusion loop" in the fusion domain (domain ii), and one or more wwihs scorepositive sequences in domain iii. with the exception of the acnpv gp , each of these proteins contains another wwihs positive domain ii sequence near the "hinge" region adjacent to the domain. herpesvirus gb proteins have an additional wwihs scale score-positive sequence in domain i. in the case of class ii and iii viral fusion proteins, the fusion loops in the fusion domain often contain sequences with positive wwihs scores. previous studies have suggested that synthetic peptides corresponding to or overlapping with sequences in viral fusion proteins that have positive wwihs scores can sometimes serve as viral entry inhibitors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, enfurvitide (fuzeon) is a -amino acid peptide that overlaps with a wwihs score-positive sequence in the transmembrane protein (tm) of hiv- , and prevents viral fusion and entry of the virus. to identify regions of hcmv gb that have a high propensity to interact with the lipid bilayer of cell membranes and which potentially may serve as hcmv entry inhibitors, we employed membrane protein explorer version . (http://blanco.biomol.uci.edu/mpex), a computer program based on the wwihs. nine sequences with significant positive wwihs scores were identified ( figure ). as expected, several of these wwihs sequences corresponded to the predicted fusion domain of hcmv gb, including the predicted fusion loops. one of the wwihs score-positive sequences spanned amino acids to (peptide - ) that had a Δg score of . kcal/mol. this sequence was split into two smaller peptides: - and - . a second large figure wimley-white interfacial hydrophobicity scale score-positive sequences in class ii and iii viral fusion proteins. sequences of a representative class ii viral fusion protein (dengue virus e) and of class iii viral fusion proteins with high potential to interface with lipid membranes (red) were identified using membrane protein explorer software (mpex version . ). as discussed in the text, a class iii domain nomenclature is used here that can apply to both class ii and iii viral fusions proteins. the alternative domain number schemes used by roche et al. and heldwein et al. are noted in parentheses. the dengue virus (denv e) stem domain sequence that was not included in the protein used to determine the crystal structure has been added. the denv stem has a positive wwihs scale score, and corresponds to a previously determined inhibitor of denv and west nile virus [ ] . segment within the fusion domain of hcmb gb had a Δg score of . , and was split, consequently, into smaller peptides: - and - . an additional peptide, - corresponding to another fusion domain sequence, was also synthesized, along with an additional peptides corresponding to other wwihs-positive domains of hcmv gb. to prevent dimer formation, cysteines were replaced with alanines. nine synthetic peptides corresponding to sequences with significant wwihs scores were synthesized and examined for their ability to inhibit hcmv infection of hff cells. peptides that were most effective are presented here ( table ) . all synthetic peptides were tested at the following concentrations: μm, μm, μm, the wwihs is a computational approach, based upon an experimentally determined algorithm to estimate the propensity of an amino acid sequence to interact with lipid membrane interfaces [ ] . using this method, we identified several regions of hcmv gb with high interfacial hydrophobicity. peptides that are analogous to several of these regions inhibited hcmv infectivity at low μm concentrations (figure , , , , and ). when tested in combination certain combinations of peptides (peptides - and - ) displayed increased inhibition of infectivity at concentrations of nm (figure ) . these results suggest that the hcmv inhibitory peptides identified here may serve as figure determination of regions within gb that display a high propensity to interact with the lipid surface of cell membranes by using wimley-white interfacial hydrophobicity scale (wwihs). wwihs identifies segments of proteins that prefer a transbilayer helix conformation to an unfolded interfacial location. we used the interface scale of the membrane protein explorer (mpex version . ) computer program to identify these particular segments of hcmv gb. the interface scale measures a residue's free energy of transfer within an unfolded polypeptide chain from water to a phosphocholine bilayer. we identified nine segments of hcmv gb that display high propensity to interact with the lipid surface of cell membrane, and designed peptides, ranging from to amino acids in length, that are analogous to the identified regions of gb. the basis for potential antiviral therapies. the success of the inhibition of fusion greatly depends, not only on biophysical properties of synthesized peptides and their concentrations, but also on the size and shape of the binding pocket of hcmv gb. it is possible that the potency of the peptides, either alone or in combination, can be increased by modifying the sequence of the peptides or by conjugating the peptide(s) to other molecules. akkarawongsa and coworkers prepared a library of overlapping peptides homologous to the ectodomain of herpes simplex virus type (hsv- ) gb- and screened for the ability of these peptides to block infection [ ] . seven out of -mer peptides inhibited infection by more than % at a concentration of μm. three peptides (gb , gb and gb ) with % effective concentrations below μm were studied further. peptide gb (residues to in hsv- gb- ) was identified as a specific entry inhibitor (ec , μm). the gb peptide (residues to in gb- ) blocked viral entry (ec ,~ μm), protected cells from infection (ec ,~ μm), and inactivated virions in solution (ec ,~ μm). of the seven inhibitory peptides identified in the akkarawongsa et al. study, three of them, corresponding to residues to (gb ), to (gb ), and to (gb ), were in regions of hsv- gb- with positive wwihs scores. the success of their study affirms our strategy of targeting the wwihs score-positive sequences as inhibitors of hcmv. two overlapping peptides spanning the residues to (gb ) and to (gb ) were not in wwihs score-positive sequence, but the analogous sequence in hcmv is wwihs score-positive. additionally, comparisons across viral families could potentiate identification of entry inhibitors. for instance, hsv- inhibitory peptide gb corresponding to domain iv, also known as a stem, is analogous to the hrobowski dengue inhibitory peptide for the class ii viral fusion proteins [ ] (figure ) . the most potential inhibitors of hcmv infection were all in domain ii. hsv- inhibitory peptide gb (residues to ) identified by akkarawongsa et al. corresponds to this region, with hcmv inhibitory peptide - being the analogous peptide ( figure ). it is possible that some of the inhibitory peptides identified for hsv- could be optimized to work for hcmv and vice versa. the fusion domain of hcmv gb may be the initial site of interaction directly with the lipids of the cell bilayer [ ] . the hcmv inhibitory peptides could competitively block these interactions, or trigger downstream fusion events, and conformational changes in gb, prematurely. inhibitory peptides corresponding to other domains likely do not block lipid interactions. inhibitory peptides may employ several mechanisms of action, and therefore those that are analogous to domain iii could block receptor interactions. as the result of this study we found that the effects of several of the hcmv inhibitory peptides did not follow a linear dose-dependent inhibition curve. this trend was also reported previously describing peptide inhibitors of dengue and west nile viruses [ ] . possibly, when the concentration of one or more peptides is too high, the peptides may self-associate, preventing them from interfering with virus infectivity. in addition to screening synthetic peptides for their ability to inhibit hcmv infection alone, we tested some peptides in combination. it is possible that peptides - and - block hcmv entry at distinct steps in the fusion process. any two peptides that work additively to block the fusion of the virion with the host cell membrane must have unique amino acid sequences, biophysical properties and be present at certain concentrations that will allow them to interact with each other, with gb and, possibly, with other glycoproteins that are instrumental in the fusion event. peptides - and - tested together showed only % inhibition at the concentration of μm each (figure ) , and did not work in an additive fashion. this result may be due to peptide-peptide interactions that do not allow interference with gb trimer formation and with necessary conformational changes of the virion required for successful fusion to occur. several drugs, including ganciclovir, its oral prodrug valganciclovir, foscarnet, cidofovir, and fomivirsen have been approved for the treatment of hcmv-associated disease. all of these drugs, with the exception of fomivirsen, have a common target, the viral dna polymerase [ ] . the above listed anti-hcmv drugs provoke not only drug-specific side effects, which include leukopenia, thrombocytopenia, anemia, bone marrow hypoplasia, diarrhea, and renal toxicity, but also the emergence of clinically relevant drug-resistant hcmv [ ] . new drugs that are more efficacious and are not toxic in treatment of hcmv infection are urgently needed. the inhibitory peptides identified here can serve as the basis for the development of a novel therapeutic against hcmv. these antiviral agents could be used as an antiviral treatment to reduce the viral load in pregnant women and neonates. it is not clear how hcmv infects the fetus during pregnancy, but some studies demonstrate that placental infection with hcmv occurs before the transmission of the virus to the fetus and suggest that the placenta plays a role in vertical transmission of hcmv from mother to fetus. also, placental viral infection has been implicated in spontaneous abortion during early pregnancy that occurs in fifteen percent of women with primary hcmv infection. placental pathology as a result of hcmv infection during pregnancy may also cause premature delivery, intrauterine growth restriction (iugr), or pre-eclampsia [ ] [ ] [ ] [ ] . enfurvitide, a -amino acid peptide also known as fuzeon works by inhibiting the structural rearrangement of hiv- gp to block the fusion of hiv- virions with their target cell membrane. brennan-benson et al. showed that fuzeon prevents vertical transmission of hiv- in pregnancy, but does not cross the placenta [ ] . we have shown that some peptides are effective at preventing hcmv infection. those lead peptides will be studied further and modified to increase their efficacy, solubility and delivery. currently available drugs to treat hcmv infection are not approved to treat pregnant women due to their potential high toxicity. hcmv infected neonates are treated with these toxic compounds only in cases of high morbidity. the antiviral peptide-based agents that may be developed as a result of this study would not require activation by virally encoded proteins, further phosphorylation by cellular enzymes or incorporation into the growing viral dna by viral dna polymerase as the current therapeutics do. such peptide therapeutics would be predicted not to provoke drug-induced resistance that is a significant problem with existing fdaapproved therapeutics to treat hcmv infection, since they employ a different mechanism of action. our cell viability assays demonstrate that the effective peptides have no statistically significant toxicity at the highest concentrations tested in our studies (data not shown). consequently, we do not expect adverse effects or toxicity due to treatments developed from these synthetic peptides. wwihs is an experimentally determined algorithm that can be used to estimate the propensity of amino acid sequences to interact with lipid membrane interfaces [ ] . sequences of hcmv gb with positive wwihs score were identified using membrane protein explorer (mpex version . ) (http://blanco.biomol.uci.edu/mpex), a computer program based on wwihs. hcmv gb synthetic peptides were synthesized by solid phase conventional n-α- -fluorenylmethyloxycarbonyl chemistry by genemed synthesis inc. (san francisco, ca). peptides were purified by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray mass spectrometry. peptide stock solutions were prepared in % dimethyl sulfoxide (dmso, spectroscopy grade): % (v/v) h o. peptide concentrations were determined by absorbance of aromatic side chains at nm (smart-spec ™ , biorad, hercules, ca). the towne strain of hcmv containing the green fluorescent protein (gfp) expression cassette was obtained from dr. daniel streblow at the oregon health science university and was propagated in human foreskin fibroblasts (hff). viral supernatants were collected days after % cpe was observed, centrifuged to clear cell debris, and filtered through a . μm filter. hff were grown in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), penicillin g ( u/ml), streptomycin ( mg/ml), and glutamax ( mm). hff were seeded at a density of . × cells in each well of a -well plate hours prior to infection. hff were washed with × dpbs and mock-or virus-infected for minutes at rt with the towne-gfp strain of hcmv ( . moi) preincubated with different concentrations of inhibitory peptides at °c for minutes. after infection, virus was removed and dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), penicillin g ( u/ml), streptomycin ( mg/ml), and glutamax ( mm) was added to each well and cells were incubated at °c for hours. gfp-positive cells were visualized hours post-infection by fluorescence microscopy and then quantified using flow cytometry. hff cells were trypsinized, centrifuged, and resuspended in % fbs dpbs. gfp-positive cells were quantified using flow cytometry (cytomics fc beckman coulter, fullerton, ca). immunobiology of human cytomegalovirus: from bench to bedside review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection primary cytomegalovirus infection in pregnancy. incidence, transmission to fetus, and clinical outcome human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments a social marketing approach to building a behavioural intervention for congenital cytomegalovirus human cytomegalovirus reinfection is associated with intrauterine transmission in a highly cytomegalovirusimmune maternal population the human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome cytomegaloviruses. in fields virology identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome disulfide bond configuration of human cytomegalovirus glycoprotein b epidermal growth factor receptor is a cellular receptor for human cytomegalovirus platelet-derived growth factor-α receptor activation is required for human cytomegalovirus infection human cytomegalovirus activates inflammatory cytokine responses via cd and toll-like receptor integrin αvβ is a coreceptor for human cytomegalovirus 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fusion human cytomegalovirus glycoprotein b is required for virus entry and cell-to-cell spread, but not for virion attachment, assembly, or egress human cytomegalovirus cell tropism and pathogenesis peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus drug targets in cytomegalovirus infection. infectious disorders-drug targets antiviral intervention, resistance and perspectives crystal structure of glycoprotein b from herpes simplex virus i structure of a trimeric variant of the epstein-barr virus glycoprotein b crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g. science the postfusion structure of baculovirus gp supports a unified view of viral fusion machines proteomics computational analyses suggest that baculovirus gp superfamily proteins are class iii penetrenes identification of the human cytomegalovirus glycoprotein b gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus structure of the prefusion form of the vesicular stomatitis virus glycoprotein g peptide inhibitors of dengue virus and west nile virus infectivity inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein fda approves fuzeon, the first fusion inhibitor model of the pre-insertion region of the spike (s ) fusion glycoprotein of the human sars coronavirus: implications for antiviral therapeutics are fusion peptides really "sided" insertional helices? cell peptides from conserved regions of paramoxovirus fusion (f) proteins are potent inhibitors of viral fusion oligopeptide inhibitors of hiv-induced syncytium formation characterization of a putative cellular receptor for hiv- transmembrane glycoprotein using synthetic peptides specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the n-terminal of the f or ha viral polypeptides curtain cc: efficacy of fusion peptide homologs in blocking cell lysis and hiv-induced fusion functional importance of the coiled-coil of the ebola virus glycoprotein a synthetic peptide from hiv- gp is a potent inhibitor of virus-mediated cell-cell fusion a synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition interaction of peptides with sequences from the newcastle disease virus fusion protein heptad repeat regions experimentally determined hydrophobicity scale for proteins at membrane interfaces multiple peptides homologous to herpes simplex virus type glycoprotein b inhibit viral infection human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis placental evidence of cytomegalovirus infection of the fetus and neonate could an infectious trigger explain the differential maternal response to shared placental pathology of preeclampsia and normotensive intrauterine growth restriction? surveillance for congenital cytomegalovirus disease: a report from the national congenital cytomegalovirus disease registry enfurvitide prevents vertical transmission of multidrugresistant hiv- in pregnancy but does not cross placenta the pymol molecular graphics system peptide inhibition of human cytomegalovirus infection the authors would like to thank dr. daniel streblow at the oregon health science university for kindly supplying the virus strain used in this study, and dr. william wimley for helpful discussions. authors' contributions lm participated in the experimental design, performed viral propagation and all experiments, and drafted the manuscript. cm and rg conceived of the study, and participated in its design and assisted lm in the interpretation of results. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -i tuhqk authors: yu, fuxun; du, yanhua; huang, xueyong; ma, hong; xu, bianli; adungo, ferdinard; hayasaka, daisuke; buerano, corazon c.; morita, kouichi title: application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of sftsv-specific human igg and igm antibodies by indirect elisa date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: i tuhqk background: severe fever with thrombocytopenia syndrome (sfts) is an emerging disease that was first reported in china in . it is caused by sfts virus (sftsv) which is a member of the phlebovirus genus in the bunyaviridae family. sftsv has been classified as a bsl pathogen. there is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. methods: the full length nucleocapsid (n) gene of sftsv yamaguchi strain was amplified by rt-pcr and cloned to an expression vector pqe . the recombinant (r) sftsv-n protein was expressed by using escherichia coli (e. coli) expression system and purified under native conditions. rsftsv-n protein based indirect igg and igm enzyme linked immunosorbent assay (elisa) systems were established to detect specific human igg and igm antibodies, respectively. one hundred fifteen serum samples from clinically suspected-sfts patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich elisa system. results: the native form of recombinant (r) sftsv-n protein was expressed and purified. application of the rsftsv-n protein based indirect igg elisa to the serum samples showed results that perfectly matched those of the total antibody sandwich elisa with a sensitivity and specificity of %. the rsftsv-n protein based indirect igm elisa missed positive samples that were detected by the total antibody sandwich elisa. the sensitivity and specificity of rsftsv-n-igm capture elisa were . and %, respectively. conclusions: the rsftsv-n protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. its preparation is simpler in comparison with that used for the total antibody sandwich system. our rsftsv-n protein-based igg and igm elisa systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. they are safe to use for diagnosis of sfts virus infection and especially fit in large-scale epidemiological investigations. severe fever with thrombocytopenia syndrome virus (sftsv), also named as fever, thrombocytopenia and leukopenia syndrome virus (ftlsv) or huaiyangshan virus, is an emergent virus that was first reported in [ ] [ ] [ ] . the sources of serum samples where the virus was identified were from patients infected in and in china. severe fever with thrombocytopenia syndrome (sfts), the disease caused by the virus has a major clinical presentations that include fever, thrombocytopenia, leukocytopenia, gastrointestinal symptoms, neurological symptoms, bleeding tendency, as well as less specific clinical manifestations [ , ] . this disease has a case-fatality rate ranging from . to % in different areas of endemicity [ ] . human-to-human transmission of sftsv was reported to occur through close contact with the blood and/or body secretions of infected patients [ ] [ ] [ ] [ ] [ ] . after the first identification of sfts, sfts cases have been reported in provinces of china [ ] . recently, the existence of this disease has also been confirmed in japan and south korea [ ] [ ] [ ] [ ] [ ] . in japan, the case-fatality rate of % ( / ) was apparently higher than that in china, where an average of % of cases was fatal [ ] . data on the high fatality rate due to sftsv indicate that sftsv is a threat to human health. another tick-borne phlebovirus, the heartland virus, which was detected in missouri, is phylogenetically associated with sftsv. it causes severe febrile illness with thrombocytopenia, leukopenia in the total blood cell count, and elevated levels of liver enzymes [ ] . for the diagnosis of sfts, laboratory confirmation is essential because the clinical manifestations of sfts are non-specific. virus isolation from the blood of viremic patients is the direct evidence of sftsv infection, however, it is time-consuming and needs high security biocontainment facility [ , ] . detection of sftsv genome could be achieved by different nucleic acid detection techniques such as reverse transcription-pcr (rt-pcr) [ , ] , real-time rt-pcr [ , ] , reverse transcriptionloop-mediated isothermal amplification assay (rt-lamp) [ ] [ ] [ ] , reverse transcription-cross-priming amplification coupled (rt-cpa) with vertical flow (vf) visualization [ ] . although these techniques have high sensitivity and specificity in early diagnosis, the duration of viraemia in sftsv infection is very short, generally - days after the disease onset [ ] . hence, the nucleic acid detecting techniques are applicable only during the acute phase of the disease which is within week after its onset. the final confirmation of infection in many cases may rely on the detection of the specific antibodies to sftsv. sftsv is a member of the phlebovirus genus in the bunyaviridae family. like other bunyaviruses, the l segment encodes the rna-dependent rna polymerase; the m segment has an open reading frame (orf) coding for a gngc precursor in the order gn-gc; whereas the s segment uses ambisense coding to express two proteins: one is a nucleocapsid (n) protein encoded by the ′ half of viral complementary sense s rna, and the other is a nonstructural (ns) protein encoded by viral sense s rna [ , , ] . nucleocapsid (n) protein is one of the most immunodominant viral proteins among members of the bunyaviridae family. recombinant n protein of rift valley fever (rvf) virus, another member of the phlebovirus genus, was reported to be used in a detection system for the laboratory diagnosis of rfv infection in humans and animals [ ] [ ] [ ] . in sftsv, jiao et al. developed a recombinant n protein based sandwich enzyme linked immunosorbent assay (elisa) for detecting the total antibodies against this virus in humans and animals [ ] . in our present report, recombinant sftsv-n (rsftsv-n) protein was expressed by using escherichia coli (e. coli) expression system and then purified. rsftsv-n protein based igg elisa and igm elisa systems were established. serum samples from clinically-suspected sfts patients were used to evaluate the newly established systems and results were compared with those obtained by using the total antibody detecting sandwich elisa system. the sftsv nucleocapsid gene encoding amino acid residues - of the full length nucleocapsid protein was successfully amplified by rt-pcr and cloned into an expression vector in frame and downstream of the sixhistidine tag (fig. a) . the sequence and reading frame of the n gene were confirmed by dna sequencing of the recombinant plasmid. the recombinant protein was successfully expressed in e. coli. most of the expressed protein was soluble ( fig. b lane ) . the rsftsv-n protein was purified from the supernatant under native conditions. analysis of purified recombinant protein by sds-page and coomassie blue staining revealed a single protein band of kd as predicted by the amino acid sequence of the nucleocapsid protein ( fig. b lane ) . the identity of the rsftsv-n protein was further confirmed by western blot assay with mouse monoclonal antibody against histidine and the use of sfts patient serum sample (fig. ) . to determine the appropriate dilutions of serum samples for the indirect igg and igm elisas using the rsftsv-n protein mentioned above, samples from healthy volunteers and sfts confirmed patients were diluted two-fold from : up to : . after the application of igg elisa to serum samples from healthy volunteers, gave an optical density (od) value of . at : and lower dilutions of the samples. at higher dilutions, : and : , the od values of all the samples were between . - . . the od values of two serum samples for sfts-confirmed patients did not change much; the value was . at : dilution and was reduced only to . at : dilution. based on these results, the dilution of serum samples for igg elisa was set at : for convenience. in igm elisa, of the healthy volunteer samples gave an od value of . in : and : dilutions but all samples gave reduced values between . - . at : dilution. the od value of two samples from sfts-confirmed patients was reduced from . at : dilution to . at : dilution. thus, the dilution for igm elisa was set at : . at the set serum dilutions, : for igg and : for igm, all the healthy volunteer serum samples gave an od values between . and . , hence, the cut-off for giving a negative result was set at . . among the serum samples from sfts-suspected patients, samples were positive by rsftsv-n proteinbased indirect igg elisa and were negative. the od value for negative samples ranged from . to . , and from . - . for positive samples. these results perfectly matched the results obtained by using the total antibody table ) . among the serum samples from sfts-suspected patients, samples were positive and were negative by rsftsv-n protein-based indirect igm elisa. the od values for negative samples ranged from . to . , and from . - . for positive samples. our indirect igm elisa failed to detect the eight samples that were positive by the total antibody sandwich elisa kit. compared with the kit, the sensitivity and specificity of the rsftsv-n-igm capture elisa were . and %, respectively ( table ). in the present study, we expressed rsftsv nucleocapsid protein in e. coli and purified the recombinant protein to near homogeneity by the his-tag based affinity chromatography under native conditions and used it as an assay antigen in the indirect igg and igm elisa. nucleocapsid protein is the most abundant protein in many viruses and recombinant nucleocapsid protein has been used for the sero-diagnosis of many viruses like, sars and nipah viruses [ , ] . in the phlebovirus family, rvf virus n protein is a good target for igg and igm elisa systems [ ] [ ] [ ] . for sfts virus, there has been only one report on the development of an elisa system (double-antigen sandwich assay system) which makes use of a recombinant n protein for detecting total antibodies and this system was validated by neutralization test [ ] . the total antibody elisa kit used in the present study was based on this system. in contrast to the preparation of the recombinant n protein used in this antigen sandwich assay system, our recombinant protein was mostly soluble and was purified under native condition without using any detergent thereby skipping the arduous work of refolding the denatured protein. the expression and purification procedures described in this study provide a simple and efficient way to obtain pure sftsv n protein in large quantity. using this rsftsv-n protein, we developed indirect igg and igm elisa for human serum, and compared with a commercial total antibody detection sandwitch elisa kit. evaluation of serum samples from sftsvsuspected patients showed a concordance rate of and . % for the igg and igm elisa in comparison with the total antibody detection sandwitch elisa kit, respectively. the sensitivity and specificity of the rsftsv-n protein based indirect igg elisa system were both % with regard to total antibody detection sandwitch elisa kit ( table ) . the sensitivity and specificity of the rsftsv-n protein based indirect igm elisa system were . % and % respectively, with regard to total antibody detection sandwitch elisa kit ( table ). the indirect igm elisa system failed to catch eight samples that were positive by the total antibody kit. this may be caused by the lower sensitivity of indirect igm elisa method or the igm antibody against sftsv did not exist in these samples. all the serum samples were collected from sfts suspected patients who recovered after their illness and all the positive samples have the specific igg (table ) . it is well known that the igg competes with the igm in binding to antigen thereby reducing the sensitivity of indirect igm elisa [ ] . still our indirect igm elisa has a sensitivity of . %; it is an acceptable level for clinical diagnosis, especially for use in developing countries. for more sensitive diagnosis method, we are currently developing an igm capture elisa system. the total antibody sandwich-elisa system which was developed by jiao et al. was widely used in china because it is simple to perform, could be used to human and all kinds of animals. but the disadvantages of this method are that it cannot distinguish igg from igm and that more volume of the serum (serum used without dilution) is required for the assay thus, limiting its application for clinical diagnosis and large scale epidemiological studies. our sftsv-n protein based systems detect igg and igm separately, so it can distinguish previous or recent infection, respectively. the serum dilution is : for igg and : for igm; this greatly save the serum used and will be quite beneficial for precious samples and large scale epidemiological studies. the rsftsv-n protein based indirect igg and igm elisa systems presented here eliminate the use of infectious virus in the antigen production, which requires high level of microbiological security facilities. hence they are safer methods for diagnosis. the expression and purification procedure for recombinant sftsv-n protein is simple and easy allowing an easy standardization of the antigen production. the advantages of using a prokaryotic host to produce recombinant sftsv-n protein would be considerable due to the ease of scale-up, and the low costs involved in growing bacteria. it would be especially useful in cases of large-scale epidemiological investigation and for application in developing countries. in conclusion, the rsftsv-n protein is highly immunoreactive in human infection and it is a good target for laboratory diagnosis. our rsftsv-n protein-based igg and igm elisa systems are safe, specific and sensitive tools for serological diagnosis of sfts virus infections and especially fit to for use in large-scale epidemiological investigations. two serum samples from sfts-confirmed patients collected in and serum samples from healthy volunteers collected in -several years before the earliest identified sfts patient was reported-were used as positive and negative controls, respectively, in determining the serum dilution for the igg and igm indirect elisa we developed in the present study. to evaluate these two assays, serum samples collected in in henan province, china were used in this study. these samples were collected from patients who recovered from an illness that was suspected to be sfts. the yamaguchi strain of sftsv (genbank accession no. ab , ab , and ab ) that was isolated in from a patient in yamaguchi prefecture, japan was inoculated to confluent monolayer of vero-e cells. these cells were then maintained at °c in eagle's minimum essential medium supplemented with % fetal calf serum and . mm of each non-essential amino acids for days. the infected culture fluid (icf) was harvested and from a μl of this icf, viral rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. the extracted rna was eluted in μl of elution buffer and then used as template for rt-pcr. rt-pcr was performed by using the primers '-ggag catgcatgtcggagtggtccagg- ' and '-aataa gcttttacaggtttctgtaagca- ' to generate the full length n gene of sftsv. sense and reverse primers contained sphi and hind iii restriction sites (underlined), respectively. the pcr amplified dna fragments were digested with sphi and hind iii, purified by a qiaex ii gel extraction kit (qiagen, hilden, germany), and subsequently cloned into the corresponding restriction site of the pqe vector (qiagen, hilden, germany). the insert of recombinant plasmid was confirmed to be in frame by dna sequencing. the expression construct encompassing amino acid (aa) - , the full length of sftsv n protein with a vector derived his-tag (histidine hexmer) at the n-terminus, was obtained. the resultant recombinant protein was designated as rsftsv-n protein. the rsftsv-n protein was expressed by inserting the recombinant plasmid containing the sftsv-n sequence into e. coli strain xl- blue and cultured at °c in luria-bertani (lb) medium containing μg/ml of ampicillin. when the optical density (od nm) of the culture reached . , the expression of recombinant protein was induced for h by the addition of . mm isopropyl β-d-thiogalactoside (iptg). after harvest by centrifugation, the e. coli pellet was washed in phosphate buffered saline solution (pbs), then resuspended in mm pbs ph . with mm nacl and frozen at − °c. after freezing and thawing three times, the cell suspension was sonicated for min with an interval of s between pulses and centrifuged at , g for min at °c. the supernatant was then applied to a talon™ imac resin column (clontech, usa). after being washed with a binding buffer ( mm pbs with mm nacl containing mm imidazole, ph . ), the purified protein was eluted with an elution buffer ( mm pbs with mm nacl containing mm imidazole, ph . ). the protein solution was aliquoted and stored in a final concentration of % glycerol at − °c until use. protein concentrations were determined by the bradford method using a bio-rad protein assay reagent kit (bio-rad, usa), and the purity of the protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). western blot analysis was performed as described before [ ] . briefly, protein marker (precision plus protein standards, bio-rad), sftsv infected vero-e cell lysates and the purified recombinant protein were separated in a - % gradient polyacrylamide gel (atto corporation, japan) before being electrotransferred onto a pvdf membrane (immobilon, millipore, usa) by using a semidry electroblotter (sartorius, germany). the membrane was blocked with blockace (yokijirushi, sapporo, japan) overnight at °c to prevent nonspecific staining, then subjected to reaction with mouse anti-histidine (ge healthcare life sciences, : dilution), or patient serum sample ( : dilution) for h at °c before incubation with rabbit anti-mouse igg, or goat antihuman peroxidase conjugate (american qualex, califonia, usa, : dilution) for h at °c. finally, the reaction was visualized by dimethyl aminobenzidine (dab) staining. serum samples from two sfts-confirmed patients and from healthy volunteers were diluted at : , : , : , : and : dilutions. the diluted sera were checked by rsftsv-n based indirect igg and igm elisa separately as described below. the appropriate dilution of the serum samples for these two assays was determined based on od values at nm. to evaluate the usefulness for diagnosis of the rsftsv-n protein we developed, we established an indirect igg and igm elisa for the laboratory diagnosis of sftsv infection in humans with the serum samples as clinical specimens. these assays have common procedures which will be described here and the procedures specific for each assay will be described under each assay. the plates used were -well nunc immunoplates (thermo scientific, denmark), and all the reagents were in μl volumes. the optimal concentration of rsftsv-n protein used to coat the microplates was determined by checkerboard titration with reference serum samples ( from sftsconfirmed patients and from healthy volunteers). the coating buffer was . m pbs, ph . , and plate coating was conducted at °c overnight. wash buffer was . m pbs with . % (vol/vol) tween (pbs-t). plates were washed three times with pbs-t after exposure to a specific reagent at each step of the procedure except at the last step as described below. dilution of all serum samples and reagents were in % nonfat milk (difco, detroit, usa) in pbs-t. incubations, except for substrate, were done for h at °c. plates with μl h o -abts substrate (kirkegaatrd & perry, gaithersburg, md) in each well were incubated for min at °c. the plates were read spectrophotometrically and od values at nm were recorded. in the indirect igg elisa, -well plates were subjected to the following steps: coating of each well with ng rsftsv-n protein per well followed by human serum samples that were diluted : , in % nonfat milk in pbs-t, then detection of bound igg with : , diluted horseradish-peroxidase-conjugated goat anti-human igg (american qualex, califonia, usa) which was made visible after adding h o -abts substrate, the last reagent in this series of procedure. od values at nm were recorded on a microplate spectrophotometer. each serum sample was tested in duplicate, and the mean od for each sample was calculated. reference serum samples were run in every assay. the mean od of a sample more than twice the mean od of the negative control serum was considered positive. the procedure for indirect igm elisa was similar to igg elisa. the changes were that the serum samples were diluted at : and the detection of bound igm was donewith : , diluted horseradish peroxidaseconjugated goat anti-human igm (american qualex, califonia, usa). serum samples from suspected sfts patients were subjected to a commercial elisa kit (xinlianxin biomedical technology co., ltd, wuxi, jiangsu, china) following the manufacturer's protocol. the kit is a double-antigen sandwich enzyme-linked immunosorbent assay kit that detects total antibodies including igg and igm against sftsv [ ] . results obtained by using this total antibody elisa kit was compared to the results obtained by applying the igg and igm indirect elisa described above. this research was approved by the institutional review board at the center for disease control and prevention of henan province. all participants gave written informed consent for the use of their serum samples for research purposes. metagenomic analysis of fever fever with thrombocytopenia associated with a novel bunyavirus in china hemorrhagic fever caused by a novel tick-borne bunyavirus in huaiyangshan, china case-fatality ratio and effectiveness of ribavirin therapy among hospitalized patients in china who had severe fever with thrombocytopenia syndrome a family cluster of infections by a newly recognized bunyavirus in eastern china, : further evidence of person-to-person transmission a cluster of cases of human-to-human transmission caused by severe fever with thrombocytopenia syndrome bunyavirus person-to-person transmission of severe fever with thrombocytopenia syndrome bunyavirus through blood contact person-to-person transmission of severe fever with thrombocytopenia syndrome virus. vector borne zoonotic dis human-to-human transmission of severe fever with thrombocytopenia syndrome bunyavirus through contact with infectious blood a highly pathogenic new bunyavirus emerged in china severe fever with thrombocytopenia syndrome: tickmediated viral disease severe fever with thrombocytopenia syndrome the first identification and retrospective study of severe fever with thrombocytopenia syndrome in japan sensitive and specific pcr systems for detection of both chinese and japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load severe fever with thrombocytopenia syndrome virus in ticks collected from humans, south korea a new phlebovirus associated with severe febrile illness in missouri early diagnosis of novel sfts bunyavirus infection by quantitative real-time rt-pcr assay development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of a new sfts bunyavirus establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of rna from the severe fever with thrombocytopenia syndrome virus detection of new bunyavirus rna by reverse transcription-loop-mediated isothermal amplification detection of severe fever with thrombocytopenia syndrome virus by reverse transcription-cross-priming amplification coupled with vertical flow visualization severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis the ecology, genetic diversity, and phylogeny of huaiyangshan virus in china cloning and expression of rift valley fever virus nucleocapsid (n) protein and evaluation of a n-protein based indirect elisa for the detection of specific igg and igm antibodies in domestic ruminants preparation and evaluation of a recombinant rift valley fever virus n protein for the detection of igg and igm antibodies in humans and animals by indirect elisa validation of an igm antibody capture elisa based on a recombinant nucleoprotein for identification of domestic ruminants infected with rift valley fever virus preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by doubleantigen sandwich enzyme-linked immunosorbent assay evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay serodiagnosis using recombinant nipah virus nucleocapsid protein expressed in escherichia coli immunoglobulin m antibody capture enzyme-linked immunosorbent assay for diagnosis of st. louis encephalitis this study was supported by a grant-in-aid for scientific research from the ministry of education, culture, sports, science and technology (mext), japan, the japan initiative for global network on infectious diseases (j-grid), mext, the authors declared they have no competing interests. key: cord- -k apj authors: fang, peng; lu, rongfei; sun, feng; lan, ying; shen, wenbiao; du, linlin; zhou, yijun; zhou, tong title: assessment of reference gene stability in rice stripe virus and rice black streaked dwarf virus infection rice by quantitative real-time pcr date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: k apj background: stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative real-time pcr (rt-qpcr), particularly for rice stripe virus (rsv) and rice black streaked dwarf virus (rbsdv) that caused seriously damage on rice plants in china and southeast asia. methods: the expression of fourteen common used reference genes of oryza sativa l. were evaluated by rt-qpcr in rsv and rbsdv infected rice plants. suitable normalization reference gene(s) were identified by genorm and normfinder algorithms. results: ubq + gapdh and ubc + actin were identified as suitable reference genes for rt-qpcr normalization under rsv and rbsdv infection, respectively. when using multiple reference genes, the expression patterns of osprib and oswrky, two virus resistance genes, were approximately similar with that reported previously. comparatively, by using single reference gene (tip -like), a weaker inducible response was observed. conclusions: we proposed that the combination of two reference genes could obtain more accurate and reliable normalization of rt-qpcr results in rsv- and rbsdv-infected plants. this work therefore sheds light on establishing a standardized rt-qpcr procedure in rsv- and rbsdv-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. rice viral diseases are major threats to rice production and have been distributed worldwide across regions depending on rice cultivation [ ] . two of the most prevalent rice viruses are rsv and rbsdv, which were transmitted by a small brown planthopper (sbph, laodelphax striatellus fallen) [ ] [ ] [ ] . when infected with rsv at the seedling stage, normally, rice plants grow poorly and often develop folded and twisted leaves, with the central leaves yellowing and withering; and plant growth may terminate and ultimately the plant will die [ ] [ ] [ ] . in china, rice stripe is very serious, especially in jiangsu province, where about . m ha per year of rice were infected by rsv during the period of to , increasing to m ha in . in heavily infected fields, rice yield is reduced by - %, and in some of the most severely infected fields, no harvest is possible [ ] . in rbsdv infected rice always develops stunted stems, dark green, twisted leaves, and white waxy swellings along veins on the abaxial surface of the leaves [ , , ] . the disease caused severely damage on rice in most parts of eastern china with due to widespread release of susceptible cultivars. since the infection damage was very severe in china and southeast asia, the understanding of the responses of rice to viral infection, especially gene expression analysis, is very important for developing strategies for disease control [ , ] . the widely used method to measure transcript abundance is rt-qpcr compared to reverse transcriptionpolymerase chain reaction (rt-pcr) and northern blot [ ] [ ] [ ] . besides being a powerful tool, rt-qpcr suffers from certain pitfalls, most important being the normalization with a reference gene [ ] [ ] [ ] . in recent years, the reference genes, such as those encoding actin (actin), tubulin (tub), glyceraldehyde- -phosphate dehydrogenase (gapdh), and s rrna, are often separately chosen for the normalization in rt-qpcr because of their constant expression levels in living organisms [ ] . nevertheless, different studies sometimes proved different or even opposing results of these reference genes, and it was demonstrated that the transcript levels of these genes actually vary under different experimental conditions [ ] [ ] [ ] [ ] . for example, different expression levels normalized by a different reference gene could be approximately folds [ ] . furthermore, myzus persicae's actin and gapdh protein were found to interaction with beet western yellows virus in vitro [ ] and some a.pisumwere's genes (actin and gapdh) considered to be potentially related to the transmission of peaenation mosaic virus and soybean dwarf virus [ ] . thus, it is important and necessary to select suitable reference gene(s) for different experimental paradigms, particularly in rsv and rbsdv infection conditions which those appropriate internal reference(s) were not identified [ ] [ ] [ ] . in this study, we reported the validation of reference genes to identify the most suitable internal control gene(s) for the normalization of rt-qpcr data upon viral infection in rice plants. using statistical algorithms genorm and norm finder [ , ] , the stability of candidate reference genes (actin, ubc, s rrna, ef- α, ubq , gapdh, α-tub, β-tub, eif- α, actin , ubq , tip -like, exp and os aoc) was examined and compared. two best reference genes were identified more stably expressed than traditional ones in rsv-and rbsdv-infected treatments. our results further indicated that the combination of these two reference genes provides a good starting point for gene expression analysis in rice viral infection plants by rt-qpcr. we characterized the phenotype of rsv-and rbsdvinfected rice plants, and the symptoms were allowed to develop under the controlled environmental conditions. rsv-infected rice developed folded and twisted leaves, with the central leaves yellowing and withering. meanwhile in rbsdv-infected rice plants, it developed stunted stems and white waxy swellings along veins on the abaxial surface of the leaves (fig. a) . rsv and rbsdv were respectively detected in inoculated rice, using rt-pcr (fig. b) . in order to evaluate the expression stability of reference genes, the candidates should be identified first. in our test, primers were designed for the fourteen commonly used reference genes, identified by blastn and tblastn searches at national center for biotechnology information (ncbi). the gene names, accession numbers, gene description and primer sequences were all provided in table . first, melting curve for four representative genes (dissociation curves for all other genes with single peak were not shown) and agarose gel analysis confirmed that each primer set gave a single amplified product of desired size ( fig. a and b) . therefore, these genes were selected for rt-qpcr validation. it is also important to accurately quantitate the quality of rna before reverse transcription. the concentration and quality of isolated rna were determined using the nanodrop spectrophotometer. agarose gel electrophoresis assay also confirmed the integrity of rna samples (fig. c) . to give an overview picture of the relative abundance of candidate reference genes, the calculated cycle threshold (ct) values were determined for each gene across all the tested rsv-and rbsdv-infected samples (fig. a) . rt-qpcr analysis indicated that candidate reference genes exhibited different levels of abundance. the ct values ranged from to , with the most lying between and . s rrna and ubq were more abundantly transcribed than others, while β-tub was the least expressed gene across all the tested samples. because the transcript levels of reference genes actually varied under different experimental conditions [ , ] , we analyzed if virus infection (rsv and rbsdv infection) altered the expression of any of the candidate genes. the ct values obtained for each gene were compared in rsvand rbsdv-infected against virus-free treatment. in this study, our results showed that each candidate reference gene expressed constantly in different treatments (fig. b) . variation in transcript quantity of rsv-and rbsdv-infected samples revealed that each tested gene approximately exhibited the similar transcript levels. therefore, above references genes were selected in the subsequent analyses. the expression stability of abovementioned candidate reference genes could be assessed by different software programs. to date, the most popular and useful method is genorm algorithm [ , ] . the genorm is a statistical algorithm which determines the gene stability measure (m) of all the genes under investigation, based on the geometric averaging of multiple control genes and mean pairwise variation of a gene from all other control genes in a given set of samples [ ] . it relies on the principle that the expression ratio of two ideal internal control genes is identical in all the samples, regardless of the experimental condition and cell-type. genes with the lowest m values have the most stable expression. in our experimental conditions, genorm analysis showed that gapdh and ubq were ranked as the best reference genes in rsv-infected plants, followed by exp, while ubc and s rrna were the least stable genes (fig. a ). when considering rbsdv-infected treatments, the average expression stability value (m) of actin and ubc were two most stable genes, followed by ubq , and those of β-tub and tip -like were the highest two (fig. b) . in general, it is not sufficient by using only one most stable reference gene to obtain an accurate and reliable result. therefore, the next question is how many reference genes should be included for rt-qpcr normalization. the genorm software also calculated the pairwise variations (v n/n+ ) between two sequential normalization factors to determine the necessity of adding further reference gene(s). as suggested by vandesompele et al. [ ] , . is a cutoff v value, below which the inclusion of an additional reference gene is not required. but this proposed value can not be taken as an absolute rule and might depend on the data. it is advisable to add additional reference genes to the normalization factor until the added gene has no significant effect [ , ] . the pairwise variation analyses showed that all of v values were less than . in this set of samples ( fig. c and d) . moreover, the v / values were higher than that of v / and v / . although v / values were much lower than that of v / and even lower values were obtained by adding more reference genes, considering practical applications, two reference genes was optimal in our experimental conditions. different algorithm methods sometimes may produce different results from the same data set. hence, all of the data were reassessed by normfinder to avoid introducing unnecessary bias. normfinder is a mathematical model which performs separate analysis of sample subgroups. it estimates intra-and inter-group variations and combines both results into a consistent value for each investigated gene [ ] . interestingly, we found that the ranking generated by this approach (fig. ) was approximately similar with those determined by genorm ( fig. a and b) . ubc and s rrna were sill ranked higher than other reference genes in rsv-infected samples. in the rbsdv-infected samples, β-tub and tip -like were also ranking the highest. although in rsv-infected samples, the rank order of exp and gapdh were slightly altered in normfinder analysis in comparison with those in genorm analyses, the stability of ubq + gapdh was high enough for reliable normalization compared to those of ubc and s rrna. also, the similar result was obtained in the rbsdvtreated samples, showing that actin + ubc were suitable for normalization in rt-qpcr analysis. in order to demonstrate the usefulness of the above validated reference genes in rt-qpcr, two important genes in virus resistant response, ospr b [ ] [ ] [ ] and oswrky [ ] [ ] [ ] [ ] , were selected and applied as target genes (fig. ) . in rsv-and rbsdv-infected samples, ubq + gapdh and ubc + actin were used as multiple reference genes, respectively. meanwhile, tip -like was used as single reference gene in our experimental conditions. as expected, in comparison with single reference gene, the normalization of ospr b using ubq + gapdh and ubc + actin , respectively, resulted in significant increase in transcript levels under virus infection conditions ( fig. a and b) . the result was similar with previous studies [ , ] . meanwhile, the expression levels of oswrky were progressively increased in the early stage of infection and then decreased ( fig. c and d) . by contrast, when tip -like was used as single reference gene, the increasing transcript patterns of ospr b and oswrky were differentially lower than those normalized by multiple reference genes. we also detect the virus gene expression in plants during viral infection. according to the results, the expression patterns of the virus gene are consistent with the expression level of virus resistant gene (fig. ) . the expression level normalized by multiple reference genes were better than those by single reference, therefore multiple reference genes were suitable for normalization in rt-qpcr analysis. rsv and rbsdv are two significant rice viruses that threat rice production. to understand the mechanism of viral transmission and discover some resistance genes, a reliable quantitation method is particularly needed. rt-qpcr is still the most commonly used technique. however, an accurate and reliable gene expression analyzed by rt-qpcr highly requires stably expressed reference genes for normalization. in fact, no one gene can act as a universal reference under different experimental conditions, and the normalization of gene expression with a single reference gene can usually lead to relatively errors fig. expression levels of candidate reference genes. a average cycle threshold (ct) values for the candidate reference genes used in this study; b comparison of the expression levels of candidate reference genes in different treatments (con: virus-free sbphs infection; rsv: rsv sbphs infection; rbsdv: rbsdv sbphs infection). -day-old rice seedlings were inoculated with or without viruliferous nymphs (rsv and rbsdv) for days. total rna was extracted from rsv-and rbsdv-infected seedlings, respectively. values were given in the form of rt-qpcr quantification cycle numbers across all tested samples. bars indicate standard error of the mean [ , ] . thus, two or more stably reference genes for normalizing are essential. in this study, the suitable reference genes for normalizing gene expression in rsv-and rbsdv-infected rice plants were identified. we first assessed the integrity of rna samples (fig. c) . meanwhile, the specificity of the rt-qpcr primer pairs was confirmed by agarose gel electrophoresis (fig. b) and melting curves analysis (fig. a) . the relative abundance of candidate reference genes was further identified (fig. ) . the results of these indicated that the candidate reference genes could be used for the subsequent analyses. to date, the commonly used methods to assess the stability of reference genes are genorm [ ] , normfinder [ ] and bestkeeper [ ] . as bestkeeper cannot test more than candidates, it was not used in this study. by using genorm and normfinder, algorithms, reference genes (table ) were evaluated. our findings revealed that ubq and gapdh were overall the most stable genes in rsv-infected rice (figs. a and a) , and actin and ubc ranked the best candidate genes under rbsdv infection (figs. b and b) . interestingly, the suitable reference genes for rsv-and rbsdv-infected rice plants were different, it is indicated that the expression of the reference genes can vary under given situations [ ] . therefore, it is vital to choose suitable reference gene(s) for normalization of gene expression. moreno et al. [ ] showed that in different cassava brown streak virus (cbsv)-infected tissues, pp a, ubq and gtpb appeared to be the most stable genes. in infected tomato plants, gapdh and ubq indicated as the most appropriate internal standards both in leaves and root tissues, and actin was also stably expressed in the infected plants [ ] . when infected with four commonly known tomato viral pathogens, act, cac and ef -α were considered as the most suitable reference genes in the studies of host-virus interactions [ ] . therefore, the selected multiple reference genes (ubq + gapdh for rsv, and ubc + actin for rbsdv) could be used for the normalization of gene expression pattern in rsv-and rbsdv-infected rice plants. normally, ef -α and s rrna were used as the reference genes in rt-qpcr experiments. previous results showed that ef -α was expressed stably in potato during biotic and abiotic stress [ ] , and s rrna was identified to be suitable for normalisation in barley yellow dwarf virus-infected cereals [ ] . in our study, the performance of above two genes was dissatisfactory. these were similar to the earlier studies in cicer arietinum and [ , ] . thus, the two traditional reference genes might not be the optimal choices for quantitating transcript level in rice during rsv-and rbsdv-infection. meanwhile, tub was widely used as reference gene for gene expression analysis. however, due to the potential regulation in various physiological states, the suitability as internal control has been questioned [ , ] . in our experimental conditions, α-tub and β-tub (in particularly) were not stable either, especially in rbsdv-infected plants. taken together, our data demonstrated that α-tub and β-tub were unsuitable for the normalization of gene expression levels in viral infected rice. to further investigate the suitability of the selected reference genes, the expression levels of two virus resistant response genes, the gene expression of ospr b and oswrky, was compared by using single and multiple reference gene(s) for normalization. in the earlier studies [ , ] , for example, the expression of ospr b gene was strongly induced by virus infection. our results further showed that when using tip -like gene for normalization, the induction patterns of ospr b and oswrky transcripts were not stronger than those using two reference genes (fig. ) . thus, the application of multiple reference genes is a better choice for gene expression analysis under virus infection situation. in conclusion, we evaluated candidate reference genes, and identified ubq + gapdh and ubc + actin as the most stably expressed reference genes in rsv-and rbsdv-infected rice, respectively. these genes will enable more accurate and reliable normalization of rt-qpcr analysis for gene expression studies to get insight into complex regulatory networks and will most probably lead to the identification of genes relevant to new biological processes, in rsv-and rbsdv-infected plants. rice (oryza sativa l. cv. nipponbare) seeds were used throughout the experiments. after the disinfection with . % hgcl for h and thorough washing with ro (reverse-osmosis) water, seeds were soaked overnight in ro water and incubation at °c for day. seedlings were then grown on plastic chambers using kimura b nutrient solution [ ] , with a / h (day/night) regimes at ± °c. rice plants infected with rsv and rbsdv, were collected from jianhu county, jiangsu province in july . young instar nymphs of sbphs were fed rsv-and rbsdv-infected rice plants for days to acquire the virus, respectively. the virus was maintained by sbphs in an insect-rearing room at a temperature of °c. virus-free sbphs were also used for the control inoculation. viruliferous or virus-free sbphs were reared on rice seedlings (oryza sativa l. cv. wuyujing no. ) in glass vessels at °c under alternating photoperiods of h of light and h of dark. -day-old seedlings were inoculated with viruliferous nymphs per plant and were kept in a growth chamber. after the incubation for days, planthoppers were removed and plants were transferred to field. virus-free sbphs were also used for control inoculation. leaf tissues were collected after , and days of virus treatment and immediately frozen in liquid nitrogen and stored at − °c until further analyses. according to previous report [ ] , rt-pcr was carried out to detect rsv and rbsdv in rice plants. primer rsrb-r ( ′-ccyatcacaaasaaatmaaaat- ′) paired with primer rsv-f ( ′-agatccagagagagtcacggaag- ′) was used to amplify a specific bp fragment for fig. the ranking of candidate reference genes based on stability values calculated by normfinder under virus-infected conditions. candidate reference genes were amplified in cdna samples from rsv-and rbsdv-infected seedlings. relative quantifications of rsv-infected (a) or rbsdv-infected (b) seedlings, were performed respectively, as described in "materials and methods" section detection of rsv. primers rsrb-r and rbsdv-f ( ′-gttcaaagacaatacactcaaaa- ′) were used to amplify a bp product, which was specific for rbsdv. preparation and quantification of dna-free total rna total rna was extracted by trizol reagent (invitrogen, gaithersburg, md, usa) according to the manufacturer's instructions. the rna was dissolved in dnase-treated distilled water. the concentration and quality of isolated rna were determined using the nanodrop spectrophotometer (thermo fisher scientific, wilmington, de, usa). only the rna samples with / ratio (an indication of protein contamination) between . and . and / ratio (an indication of reagent contamination) greater than . , were used for the analyses [ , ] . the integrity of rna samples was assessed by agarose gel electrophoresis. all rna samples were adjusted to the same concentration and measured again to homogenize rna for the subsequent experiments. reverse transcription and quantitative real-time pcr assay cdna was synthesized from μg of total rna using an oligo (dt) primer and m-mlv reverse transcriptase (bioteke, beijing, china). rt-qpcr was performed using the ssofast tm eva green® supermix (bio-rad) with the bio-rad iq rt-qpcr system. combined with all internal control genes used recently [ , , ] , we further selected genes as the candidate reference genes in this study (table ) . additionally, the specific primers ′-acgccttcacggtccatac- ′ and ′-aaacagaaagaaacagagggagtac- ′ were used for ospr b (ak ); ′-tcagtggagaagcgg gtggtg- ′ and ′-gggtggttgtgctcgaag gag- ′ were used for oswrky (ef ). the efficiency and specificity of all the primers were checked by melting curve analysis, similar to previous report [ ] . using the rsv cp, sp gene and rbsdv p - , p - gene, the dynamics of viral infection in rice plants was measured by reverse-transcription real-time pcr. the primer and products were shown in table . each quantitative real-time pcr was performed using . μl cdna, μl of ssofast tm eva green® supermix and . μm forward and reverse primers were used in a total volume of μl. all tubes were subjected to denaturation for min at °c, followed by cycles of °c for s, and °c for s. sybr green absorbance was detected at °c. all reactions were conducted in triplicate. amplicon dissociation curves (melting curves), were recorded after cycle by heating from °c to °c at a ramp speed of . °c ⁄ min. fig. relative expression levels of ospr b and oswrky using single or multiple reference gene(s) for normalization during rsv-(a and c) and rbsdv-(b and d) infection. -day-old rice seedlings were inoculated with viruliferous nymphs (rsv and rbsdv) for days. total rna was extracted from rsv-and rbsdv-infected seedlings, respectively. tip -like was used as a single reference gene, while ubq + gapdh and ubc + actin were used multiple reference genes under rsv-and rbsdv-infection plants, respectively, in our experimental conditions. bars indicate standard error of the mean the expression stability of the candidate reference genes were analyzed by using genorm [ ] and normfinder [ ] . expression levels were assessed based on the number of amplification cycles needed to reach a specific threshold (cycle threshold; ct) in the exponential phase of rt-qpcr. for both programs, raw ct values of each gene were converted into relative quantities before inputting into software. the relative expression levels of corresponding genes were calculated relative to the maximum abundance in different samples. the highest relative expression for each gene was set to . . the genorm fig. relative expression levels of virus genes (cp and sp for rsv, p - and p - for rbsdv) using single or multiple reference gene(s) for normalization during rsv-(a and c) and rbsdv-(b and d) infection. -day-old rice seedlings were inoculated with viruliferous nymphs (rsv and rbsdv) for days. total rna was extracted from rsv-and rbsdv-infected seedlings, respectively. ubq + gapdh and ubc + actin were used multiple reference genes under rsv-and rbsdv-infection plants, respectively [ ] derives a stability measure (m). via a stepwise exclusion of the least stable gene, it creates a stability ranking. it also estimates the number of genes required to calculate a robust normalization factors, and performs a stepwise analysis (more stable to less stable genes) to calculate the pairwise variation (v n/n+ ) between two sequential normalization factors containing an increasing number of genes. normfinder algorithm [ ] used an anova-based model to estimate intra-and inter-group variation. it combines these results to provide a direct measurement of the variation in the expression for each gene [ ] . statistical significance of ct differences between treatments was calculated by the mann-whitney t test using the graphpad prism software. additional file : figure s . relative expression levels of ospr b and oswrky using single or multiple reference gene(s) for normalization during rsv-(a and c) and rbsdv-(b and d) infection. -day-old rice seedlings were inoculated with viruliferous nymphs (rsv and rbsdv) for days. total rna was extracted from rsv-and rbsdv-infected seedlings, respectively. in our experimental conditions, ubq + gapdh and ubc + actin were used multiple reference genes under rsv-and rbsdv-infection plants. 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accurate transcript normalization in citrus genotypes under different experimental conditions role of root hairs and lateral roots in silicon uptake by rice a simplified method for simultaneous detection of rice stripe virus and rice black-streaked dwarf virus in insect vector quantification of mrna using real-time rt-pcr reference genes for quantitative real-time pcr analysis in the model plant foxtail millet (setaria italica l.) subjected to abiotic stress conditions submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was financially supported by grants from the national key basic research and development program ( program) of china ( cba ), the national natural science foundation ( ), the special fund for agro-scientific research in the public interest ( ), the jiangsu agricultural scientific self-innovation fund (no.cx [ ] ) and the jiangsu province science and technology support project (be ). the authors declare that they have no competing interests. key: cord- -tofch j authors: bai, juan; chen, xinhui; jiang, kangfu; zeshan, basit; jiang, ping title: identification of vp peptides diagnostic of encephalomyocarditis virus from swine date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: tofch j background: encephalomyocarditis virus (emcv) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in china. emcv vp protein was one of the most important structural proteins and played an important role in the protective immunity. in this study, monoclonal antibodies (mcabs) against emcv vp were screened and identified. results: epitope mapping results indicated that mcabs ( e , a , c ) specifically recognized the linear epitopes v( )enaek( ), mcabs ( d , a , a , a , g ) recognized the epitope f( )vaqpvy( ), and mcabs g and a recognized p( )igaftvk( ). protein sequence alignment of vp with emcv isolates indicated that the epitope f( )vaqpvy( ) was conserved in all the reference strains. the epitopes p( )igaftvk( ) and v( )enaek( ) only had or variable amino acid among the reference strains. the d model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle. conclusions: in this study, ten mcabs against emcv vp were developed and three b-cells epitopes ( - aa, - aa and - aa) were defined in vp . all the results herein will promote the future investigations into the function of vp of emcv and development of diagnostic methods of emcv. encephalomyocarditis virus (emcv) is characterized by not only myocarditis and encephalitis, but also neurological diseases, reproductive disorders and diabetes in many mammalian species [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . rodents are considered to be natural hosts of emcv and are thought to be the primary reservoir and disseminators of the virus. out of all domestic animals, pigs are the most susceptible to emcv infection, which can cause severe economic losses on pig production due to high mortality in piglets as a result of respiratory failure [ , , ] and in sows as a result of myocarditis and reproductive failure [ ] [ ] [ ] . emcv is a member of the cardiovirus genus of picornaviridae [ ] and has a single-stranded positive-sense rna of approximately . kb [ ] . the orf encodes for a polyprotein that comprises both non-structural and structural elements divided into three primary precursor molecules, namely p , p and p , encoding for distinct proteins. the structural proteins vp , vp , vp and vp make up the viral capsid and are encoded in the p region towards the ′-end of the genome. the viral capsid proteins, in their capacity to interact with cellular receptors, are crucial for this entry step and may be considered to be factors that can modulate emcv virulence [ ] . the three major capsid proteins, vp , vp and vp that constitute the external virion shell of picornaviruses, are considered to play a pivotal role in viral infection and host recognition [ ] . among them, vp is one of the most antigenic and can stimulate the organism to produce neutralization antibody [ , ] . the detailed analysis of epitopes is important for the understanding of immunological events and for the development of epitope-based marker vaccines and diagnostic tools for various diseases [ ] [ ] [ ] . in this paper, vp protein of emcv nj strain was expressed by the escherichia coli system and ten monoclonal antibodies (mcabs) against the recombinant emcv vp were screened and identified. three linear epitopes ( - aa, - aa and - aa) were identified in vp protein of emcv. after screening of the fusion cells by indirect elisa, the positive hybridoma cells secreting the antibodies against vp protein were selected and sub-cloned thrice by limiting dilution, and ten mcabs were generated and named as d , f , g , d , a , a , a , g , e , a and c . the results of western blot showed that these mcabs were all directed against the purified emcv and rvp protein expressed in e. coli ( figure ). meanwhile, ifa results also showed that these mcabs could react with the emcv in bhk cells (figure ). the titres of antibodies in the cells cultures were : - . twenty-six overlapping vp protein gene fragments were prepared by pcr and cloned into a gst fusion protein expression vector. after validation by restriction analysis and nucleotide sequencing, recombinant proteins encoded by each of these constructs were expressed in e.coli. the resulting recombinant proteins were identified using western blot with gst-tagged monoclonal antibody. the results showed that all the proteins were reactive with gst-tagged mcab ( figure ). in order to map the minimal sequences of the epitopes recognized by mcabs, the series of truncated recombinant proteins were used in western blot was to identify the reactivity of each of anti-vp mcabs. the results showed that mcabs ( e , a and c ) could reacted with fragments (f , f and f ) containing the six amino acids v( )enaek( ), but not reacted with the fragments with the deletion of any one of the six amino acids (f , f and f ). it indicated that amino acids v( ) enaek( ) in vp were essential for this epitope ( figure a ). in the same way, mcabs ( d , a , a , a and g ) reacted against the epitope comprising f ( )vaqpvy ( ) , because they could react with the fragments f , f , f and f , but not f , f and f ( figure b ). mcabs ( g and a ) could reacted with fragments (f , f , f and f ) containing the eight amino acids p( )igaftvk( ), but not reacted with the fragments with the deletion of any one of the eight amino acids (f , f and f ) ( figure c ). meanwhile, those purified truncated fragment proteins were used as the antigens in elisa to detect the levels of those mcabs. the results showed that the od values of mcabs ( e , a and c ) in f , f , f and f groups were significantly higher than those in f , f , f and f (p < . ) ( figure a ). the od values of mcabs ( d , a , a , a and g ) in f , f , f , f , f and f groups were markedly higher than those in f , f and f groups (p < . ) ( figure b ). the levels of mcabs ( g and a ) in the groups of f , f , f and f were notablely higher than those in f , f and f (p < . ) ( figure c ). the synthetic peptides (p - aa, p - aa and p - aa), purified rvp and gst- - aa (f ) proteins were used as elisa antigens. the results showed that od values of e , a and c reacted with p - aa was highest in those mcabs. and the levels of mcabs ( d , a , a , a and g ) reacted with p - aa were obviously higher than those of other mcabs (p < . ). mcabs g and a reacted with p - aa were significantly higher than other mcabs (p < . ). it indicated that the synthetic peptides p - aa, p - aa and p - aa have immune reactivity with the pepide specific mcabs, respectively ( figure ). however, the od values of anti-emcv mice serum were relatively lower than those of mcabs reacted with p - aa, p - aa and p - aa. it suggested that these synthetic peptides reacted weakly with emcv-specific serum by peptide-based elisa ( figure ). alignment of the amino acid sequences of the different emcv isolates revealed that the minimal epitope f( ) vaqpvy( ) was highly conserved among all the reference emcv strains, the epitope v( )enaek( ) was relatively conserved among all emcv strains, except for a k → r change in gxlc, d variant, emc-b and emc-d strains. in addition, the epitope p( )igaftvk ( ) was conserved among the strains isolated from china. whereas there were variable amino acids in different site of this region among the reference strains isolated from other countries ( figure ). there existed a very close structural analog to nj in structures published in and with pdb accession numbers mev and mec respectively both for the mengo virus. the vp protein of nj had very few amino acid changes and no deletions or additions in the sequence compared to the mengo virus vp sequence as shown in figure . therefore any of the structures could be used a d model to map the epitopes within the context of the complete viral particle. as show in figure , the epitopes presented as spheres were shown within the context of the complete particle with colors of red for vp , green for vp , yellow for vp and blue for vp . the epitopes v( )enaek( ) and f ( ) vaqpvy ( ) shown as cyan and magenta spheres were partially buried in the viron. p( )igaftvk( ) shown as orange spheres was located circa the -fold icosahedral axis of symmetry and was on the surface. b cell epitopes are linear or conformational resulting from unique protein folding. they could be defined as regions on the surface of the native antigen that could bind to b-cell receptors or specific antibodies [ , [ ] [ ] [ ] . in this study, ten monoclonal antibodies (mcabs) against the vp of emcv were prepared by inoculated with the purified killed emcv, and then three minimal epitopes [v ( )enaek( ), f( )vaqpvy ( ) , and p( )igaftvk ( )] were identified in n terminus of the emcv vp , using these mcabs and a series of recombinant truncated vp . vp is one of the most important structures proteins of emcv, which can stimulate the organism to produce neutralization antibody [ ] . immunization of mice with recombinant vp protein (plasmids or recombinant adenoviruses expressing small hairpin rnas targeted to vp protein genes of emcv), which could provide protective efficacy against emcv [ , ] . dna vaccines encoding an emcv-d vp antigen confer protective immunity against heterologous challenge with emcv-k, indicating that vp is a potential vaccine candidate for controlling emcv infection [ ] . thus it is need to explore the epitopes related to the protection or diagnosis of this disease, even thought the residue at position , and are related to neutralization epitopes. in this study, the ten anti-vp mcabs all did not have the ability to neutralize the emcv in cells cultures (data not shown here). however, they all could react with the emcv antigen in western blot and ifa and could be used for diagnosis of this virus. sequence analysis of both variants revealed one amino acid exchange within the capsid protein vp , demonstrated that the diabetogenic and non-diabetogenic emcv variants differing in only one single amino acid [ ] [ ] [ ] [ ] . studies of theiler's murine encephalomyelitis virus (tmev) demonstrated that the amino acids on the loops exposed to the surface of the virion were important disease determinants [ ] . the residue was located in the loop connecting the βb and βc strands of vp , which was exposed at the surface of the capsid and was known to be part of a neutralization epitope [ ] . in this study, the results of alignment of the amino acid sequences of different emcv isolates revealed that the epitope f( )vaqpvy ( ) was highly conserved among all the reference emcv strains. there were only or amino acid residue different in the epitopes p( )igaftvk( ) and v( ) enaek( ) between those strains. according to the model of mengo virus, the d model of vp of emcv was also analyzed. in the picture, p( )igaftvk( ) shown as orange spheres was located circa the -fold icosahedral axis of symmetry and was on the surface. meanwhile, v( ) enaek( ) and f( )vaqpvy ( ) were at the n-terminus and located *under* the vp protein and sandwiched with the vp protein inside the particle. the obtain of the mc-abs against these two epitopes in this study might be explanted by the "breathing" of the n-terminal tail between the vp and vp proteins. these two epitopes could be used for diagnosis of this virus because of the results of alignment that these two regions were highly conserved among all the reference emcv strains. here, ten mcabs against emcv vp were developed and three b-cells epitopes ( - aa, - aa and - aa) were defined in vp . the results may contribute to understand the antigenic structure of vp protein deeply and facilitate the development of diagnostic method of emcv. emcv isolate nj (genbank accession no. hm ) used in this study was isolated and kept in our lab (submitted -jul- ). bhk- cells were kept in dulbecco's modified eagle medium supplemented with % fetal bovine serum. the sp / cells were grown in rpmi medium supplemented with % fetal bovine serum. female balb/c mice, - weeks of age, were purchased from the laboratory animal centre of nantong university. hat and ht media supplement ( ×) were purchased from sigma. horseradish peroxidase (hrp)-tagged goat anti-mouse igg was purchased from wuhan boster biological technology co. rapid elisa mouse mcab isotyping kit was purchased from pierce. the vp gene was amplified by pcr using the specific primers kept in our lab. pcr products were digested by bamh i and ecor i, and cloned into prokaryotic expression vector pet- a(+) subsequently. the recombinant plasmids were verified by analysis and nucleotide sequencing, and then transformed into e.coli bl cells for fusion proteins expression. the transformants were cultured at °c in luria-bertani broth containing . mg/ml kanamycin until the optical density at nm (od ) reached . . the cells were incubated for another h in the presence of . mm isopropyl-β-d-thiogalactoside and harvested by centrifugation. the e. coli/pet- a-vp transformants were lysed by sonication, and then centrifuged at g for min at °c. the proteins were purified by probondtm purification system, according to the manufacturer's instructions (invitrogen). the purified recombinant vp (rvp ) protein was detected by sds-page and western blot, and the concentration was determined by nanodrop spectrophotometer (thermo). emcv was sedimented and purified from the culture medium with peg- by ultracentrifugation. the purified emcv were analyzed by sds-page and western blot with anti-emcv sera. the mcabs were developed and obtained as follows. briefly, inactivated emcv was combined with equal volumes of complete/incomplete freund's adjuvant (sigma-aldrich) at final concentration of . mg/ml. each female balb/c mice ( -week-old) was immunized with . mg emulsion at weeks intervals for weeks. three days after the last injection, the spleens were surgically removed from the mice and fused with mouse myeloma cells (sp / ) using % (v/v) peg. the fused hybridoma clones were screened by the indirect elisa for mcabs that have strong reactivity with the rvp but not his protein or bound emcv but not bhk- cells' protein. selected clones were subcloned by limiting dilution. ascites fluids were induced in pristine-primed balb/c mice. finally, the mcabs were identified by western blot and indirect immunofluorescence assay (ifa). for screening the mcabs, elisa plates were coated with the purified rvp protein and e.coli/pet- a lysate (as the negative control) at the concentration of . mg/ ml in buffer bicarbonate (ph . ) at °c overnight or coated with the purified emcv and bhk- cells' protein at the concentration of . mg/ml as the same way above. after washing with pbs containing . % tween- (pbst) three times, the plates were blocked figure the locations of the three epitopes. the picture was about the complete particle of mengo virus. vp is shown in red ribbon, vp is shown in green ribbon, vp is shown in yellow ribbon, and vp is shown in blue ribbon. the epitopes presented as spheres, v( )enaek ( ) is colored cyan, f( )vaqpvy( ) is colored magenta, and p( )igaftvk( ) is orange. the figure was generated using ucsf chimera [ ] . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). with % non-fat milk in pbst at °c for h. then wells were incubated with the supernatant of hybridoma at °c for h. the sera isolated from immunized mice and the supernatant of sp / cells were used as positive and negative controls, respectively. after three washes with pbst, wells were incubated with a : , diluted goat antimouse igg-hrp at °c for h. after washing, the plates were incubated with substrate solution tetramethyl benzidine (tmb) at °c for min, and the reaction was stopped with m h so in each well ( ml/well).the od was read at nm in an automatic elisa plate reader. the criteria were judged as follows: the od of the positive control should be more than . , the od of the negative controls should be lower than . . the results were expressed as the ratio of od produced by the samples compared to the negative control. samples, giving a ratio value higher than . , were considered to be positive. for mapping the epitopes precisely, a series of truncated fragments of protein were expressed in e.coli. the epitopes recognized by mcabs were mapped by truncated vp fragments based on indirect elisa as described above. in order to identify the minimal antigenic epitopes recognized by mcabs, the vp gene was divided into truncated fragments (figure ). a series of primers (table ) were synthesized to amply the different truncated fragments of vp protein. pcr products were separately cloned into the xho i and bamh i sites of pgex- p- expression vector. the recombinant proteins were expressed in e.coli bl and identified by sds-page and western blot with the gst-tagged monoclonal antibody (boshide, wuhan, china), and then were used to determine the epitopes recognized by mcabs using indirect elisa and western blot methods. to identify the proteins expressed in e. coli (vp and truncated vp proteins), the proteins were separated by sds-page ( - %) and transferred to nitrocellulose membrane (pall corporation). the mcabs (dilution of : - : ) bound to the proteins on the membrane was detected by goat anti-mouse igg-hrp (boshide, wuhan, china) at °c for h. detection was performed using chemiluminescenceluminol reagents (supersignal west pico trial kit, pierce). bhk cells infected with emcv nj strain in -well culture plate were rinsed with pbs and fixed with cold ethanol for min at °c. the cells were washed, and three linear epitopes (p - aa, p - aa and - aa) identified in this study were synthesized using fmoc solidphase chemistry (invitrogen) with > % purity. they were coated in reacti-bind™ amine-binding, maleic anhydride -well plates (pierce, u.s.a.) at the concentration of ug/ml for h at room temperature. meanwhile, purified gst- - aa (f ) and rvp protein were used as positive antigens control. mice anti-emcv serum (at dilution of : ) were added as the primary antibody and incubated at °c for h. the spf mice serum was used as negative antibody control. the following detection steps were the same as described above. nucleotide sequences of emcv strains from different countries were retrieved from genbank. the amino acid sequences of identified b-cell epitopes were aligned using dnastar megaligen software. the representative emcv strains were listed in table . molecular graphics and analyses were performed with the ucsf chimera package. chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p -gm ). the differences in the level of antibodies between different groups were determined by one-way repeated measurement anova and least significance difference (lsd). differences were considered statistically significant when p < . . the encephalomyocarditis virus pathogenesis of encephalomyocarditis experimental infection in young piglets: a potential animal model to study viral myocarditis an outbreak of encephalomyocarditis-virus infection in free-ranging african elephants in the kruger national park an encephalomyocarditis virus epizootic in a baboon colony fatal inflammatory heart disease in a bonobo (pan paniscus) screening for antibodies against zoonotic agents among employees of the zoological garden of vienna a wild-type porcine encephalomyocarditis virus containing a short poly(c) tract is pathogenic to mice, pigs, and cynomolgus macaques encephalomyocarditis virus infections in an australian zoo persistence of encephalomyocarditis virus (emcv) infection in piglets genetic variability of encephalomyocarditis virus (emcv) isolates outbreaks in quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus reproductive failure in sows following experimental-infection with a belgian emcv isolate serological survey on prevalence of antibodies to avian paramyxovirus serotype in china retrospective genetic characterisation of encephalomyocarditis viruses from african elephant and swine recovers two distinct lineages in south africa the nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region host and virus determinants of picornavirus pathogenesis and tropism characterization of mengo-virus neutralization epitopes. . infection of mice with an attenuated virus protective immunity against heterologous challenge with encephalomyocarditis virus by vp dna vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene identification of immunodominant b-and t-cell combined epitopes in outer membrane lipoproteins lipl and lipl of leptospira interrogans identification of a conserved linear b-cell epitope at the n-terminus of the e glycoprotein of classical swine fever virus by phage-displayed random peptide library identification of b-cell epitopes in the nsp protein of porcine reproductive and respiratory syndrome virus ucsf chimera-a visualization system for exploratory research and analysis identification of continuous b-cell epitopes on the protein moiety of the -kilodalton cell wall mannoprotein of candida albicans belonging to a family of immunodominant fungal antigens identification of a linear b-cell epitope on avian reovirus protein sigmac selection of sars-coronavirus-specific b cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice inhibition of encephalomyocarditis virus replication by shrna targeting d and ab genes in vitro and in vivo protective efficacy of adenovirus-mediated small interfering rnas against encephalomyocarditis virus challenge in mice enhancement of vp -specific immune responses and protection against emcv-k challenge by co-delivery of il- dna with vp dna vaccine molecular analysis of the capsid coding region of a virulent encephalomyocarditis virus isolate after serial cell passages and assessment of its virulence gain or loss of diabetogenicity resulting from a single point mutation in recombinant encephalomyocarditis virus analysis of sequence and pathogenic properties of two variants of encephalomyocarditis virus differing in a single amino acid in vp alteration of encephalomyocarditis virus pathogenicity due to a mutation at position of vp mutations that affect the tropism of da and gdvii strains of theiler's virus in vitro influence sialic acid binding and pathogenicity efficient infection of buffalo rat liver-resistant cells by encephalomyocarditis virus requires binding to cell surface sialic acids this work was supported by grants from the jiangsu natural science foundation to young researcher (bk ), the china agricultural research system foundation (cars- ), the special fund for agroscientific research in the public interest ( - ), the national natural science foundation ( ), and the priority academic program development of jiangsu higher education institutions (papd). the authors declare that they have no competing interests. key: cord- -j t qu authors: uccellini, lorenzo; ossiboff, robert j; de matos, ricardo ec; morrisey, james k; petrosov, alexandra; navarrete-macias, isamara; jain, komal; hicks, allison l; buckles, elizabeth l; tokarz, rafal; mcaloose, denise; lipkin, walter ian title: identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (python regius) date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: j t qu background: respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified. findings: pneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (python regius). eight of snakes had evidence of bacterial pneumonia. high-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. pcr indicated the presence of viral rna in lung, trachea, esophagus, liver, and spleen. in situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. phylogenetic analysis based on a , amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily torovirinae. conclusions: this report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses. nidovirales is a large order of positive sense, singlestranded rna (ssrna) viruses that consists of the many genera and species in the families coronaviridae, arteriviridae, roniviridae and mesoniviridae. although the genomes of nidoviruses vary in length, ranging from to kilobases (kb), the organization of the genomes are similar across the entire order [ ] [ ] [ ] [ ] . the ′ end of the genome encodes two replicase polyproteins (pp a and pp ab), structural proteins and accessory proteins. genes downstream of the replicase polyprotein gene are expressed from a nested set of ′-coterminal subgenomic mrnas, a replication strategy unique to the nidovirales [ ] [ ] [ ] . nidoviruses infect a broad range of hosts including humans and other mammals, birds, fish, insects and crustaceans [ ] [ ] [ ] [ ] . although reptiles are susceptible to infection by a wide variety of viruses (as reviewed in [ ] ), nidovirus infections have not previously been described. viruses affecting the reptile respiratory tract include herpesviruses [ ] , iridoviruses [ ] , adenoviruses [ ] , flaviviruses [ ] , and, of particular importance in snakes, paramyxoviruses [ ] and reoviruses [ ] . here we report the discovery of a novel nidovirus in a collection of ball pythons (python regius) in upstate new york with pneumonia, tracheitis and esophagitis. the snakes were found dead between july and september . gross postmortem examination was performed on snakes. snake , a -year-old, female piebald color morph ball python, was submitted in july of . snake , a -year-old, male lesser platinum color morph ball python, was submitted in december of . snake , a -year-old female paradox color morph ball python, and snake , a -year-old female pastel color morph ball python, were submitted in september . after gross evaluation, samples of tissues were collected and saved in % neutral buffered formalin, routinely processed and mounted in paraffin. five μm paraffin ribbons were cut and stained with either hematoxylin and eosin (h&e) or gram's stain for histologic examination. eleven tissues, including lung (n = ), liver (n = ), spleen (n = ) and esophagus (n = ) from the snakes were collected and stored at − °c. gross and histologic findings in all four snakes were primarily restricted to the respiratory and upper gastrointestinal tracts ( table ). hematoxylin and eosin stained sections ( figure a through f) revealed marked hyperplasia of epithelial cells lining air exchange areas (pneumocytes) with significant mononuclear (lymphocytes and plasma cells) and granulocytic (heterophils) interstitial inflammation and epithelial necrosis ( figure b ). similar inflammatory and hyperplastic changes were also present in the trachea ( figure d ), esophagus ( figure f ) and oral cavity. gram-negative stained bacteria are shown in lung tissue from a snake with bacterial bronchopneumonia ( figure h ). total nucleic acids were extracted from snake samples (lung and spleen for snake , lung, spleen and esophagus for snake , spleen and liver for snake , lung and trachea for snake ) using the easymag (biomérieux, inc.) platform; samples from snakes and were depleted of ribosomal rna (ribo-zero™ rrna removal, epibio) and treated with dnase i (turbo dna-free™, ambion). cdna synthesis was performed using superscript ii first-strand synthesis supermix (invitrogen). viral discovery was performed using broadly reactive consensus pcr assays targeting common respiratory viruses of animals, including paramyxoviruses [ ] [ ] [ ] , reoviruses [ ] and caliciviruses [ ] . when pcr analysis failed to yield a causative agent, high-throughput sequencing was performed on all samples originating from snakes and (ion pgm, life sciences). on average, , reads were obtained from each sample. all reads were processed by trimming primers and adaptors, length filtering, and masking of low-complexity regions (wu-blast . ). to remove host sequences, the remaining reads were subjected to a homology search using blastn against a database consisting of ribosomal and genomic metazoan sequences. following the processing, an average of , reads per sample remained for further analysis. nucleotide sequence analysis (blastn) of processed reads was uninformative; however, amino acid analysis (blastx) revealed multiple reads with amino acid homology of < % to the polyprotein region of the nidovirales subfamily torovirinae, including breda virus, white bream virus, and fathead minnow virus. assembly of all torovirinae-like reads generated a , nt contig with % amino acid homology to the replicase polyprotein ab of fathead minnow virus. the presence of this , nt sequence in both snakes was confirmed by pcr using primers shown in table . cycling conditions are described in a footnote to table . samples from multiple tissues of snakes and were also screened and tested positive for this virus. for phylogenetic analysis, the , nt sequence was translated with se-al v . a , and a , amino acid fragment was aligned against all nidovirales sequences from genbank using clustalw. the best-fit model of amino acid substitution, the whelan and goldman (wag) matrix, was selected using the maximum likelihood method implemented in mega version . [ ] . a bootstrap-supported ( replicates) maximum likelihood phylogenetic tree was constructed using mega version . . the ball python-associated virus clustered within the torovirinae subfamily ( figure ) . a neighbor-joining phylogenetic method was also implemented with congruent results. based on the phylogenetic position and the genetic distances between in situ hybridization to a nt fragment of the genomic polyprotein ab region was used to assess viral infection and distribution in the lung tissue. positive cytoplasmic staining, consistent with the presence of the lungs from infected snakes (b) were characterized by marked pneumocyte hyperplasia and mixed mononuclear and granulocytic inflammation compared to the uninfected snake (a). the normally thin and ciliated tracheal mucosa (c) was severely thickened in infected snakes (d) with epithelial necrosis, loss of the ciliated mucosal border and moderate mixed inflammation. the esophageal mucosa that is normally rich with mucus-producing epithelial cells (e) was also severely hyperplastic in infected snakes, with necrosis and mixed inflammatory infiltrates similar to those seen in the trachea (f). gram-negative bacteria are present in snake with bronchopneumonia (h) but not in snake without bronchopneumonia (g). viral nucleic acid, was confirmed in the cytoplasm of pulmonary cells, presumably epithelial cells ( figure a ). the specificity of probes for in situ hybridization was confirmed by the absence of signal when the same probe was used on control pulmonary tissue from an uninfected, -year-old, female ball python maintained at college of veterinary medicine, cornell university ( figure b ). respiratory disease can be an important cause of morbidity and mortality in both wild and captive reptiles. in captivity, reptiles, and particularly snakes, are frequently maintained in collections with a high population density in relatively small spaces. as such, disease transmission within collections can occur rapidly, and early detection and diagnosis is critical in controlling disease spread. although of snakes with disease in the collection showed gram-negative rods by gram stain and follow up culture in revealed the presence of aeromonas sp., pseudomonas sp., serratia sp., no evidence of bacterial infection was found in snakes. in contrast, all snakes with epithelial hyperplasia in the trachea, lung and esophagus and mononuclear inflammatory infiltrates had viral signal by pcr and ish. in concert, these data suggest a role for this nidovirus in the pathogenesis of respiratory disease. however, unequivocal implication will require experimental infection studies. the identification of this novel nidovirus expands our understanding of nidoviral diversity and provides insight into the pathogenesis of respiratory disease in snakes. phylogenetic analysis indicated that the virus belongs to a novel genus within the torovirinae subfamily distinct from the torovirus and recently characterized banifivirus genera [ ] . due to overlapping clinical signs and pathologic lesions of the newly discovered nidovirus with the best characterized viral respiratory pathogens of snakes, paramyxoviruses and reoviruses [ , ] , it is possible that nidoviral infections were previously misdiagnosed or overlooked. pcr-based detection methods to rapidly determine infection status and etiology of respiratory disease in snakes are recommended to guide decisions for managing husbandry and veterinary care. the data set supporting the results of this article is included within the article. nidovirales: a new order comprising coronaviridae and arteriviridae gill-associated virus of penaeus monodon prawns: an invertebrate virus with orf a and orf b genes related to arteri-and coronaviruses mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses the coronaviruslike superfamily gill-associated nidovirus of penaeus monodon prawns transcribes -coterminal subgenomic mrnas that do not possess -leader sequences nidovirus transcription: how to make sense…? rna transcription analysis and completion of the genome sequence of yellow head nidovirus in virus taxonomy: classification and nomenclature of viruses. eighth report of the international committee for taxonomy of viruses an introduction to nidoviruses in vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled rna, double-stranded dna probes and oligonucleotides by in situ hybridization an insect nidovirus emerging from a primary tropical rainforest viruses infecting reptiles. viruses conjunctivitis, tracheitis, and pneumonia associated with herpesvirus infection in green sea turtles respiratory and pharyngo-esophageal iridovirus infection in a gopher tortoise (gopherus polyphemus) adeno-like virus in esophageal and tracheal mucosa of a jackson's chameleon (chamaeleo jacksoni) from viruses and viral diseases of reptiles pulmonary lesions in experimental ophidian paramyxovirus pneumonia of aruba island rattlesnakes, crotalus unicolor isolation and experimental transmission of a reovirus pathogenic in ratsnakes (elaphe species) immunoperoxidase detection of ophidian paramyxovirus in snake lung using a polyclonal antibody immunohistochemical detection of ophidian paramyxovirus in snakes in the canary islands molecular diagnosis of paramyxovirus infection in snakes using reverse transcriptase-polymerase chain reaction and complementary deoxyribonucleic acid:ribonucleic acid in situ hybridisation feline calicivirus mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods genetic analysis of a novel nidovirus from fathead minnows identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (python regius) the authors would like to thank the following: simon williams; raja duraisamy; simon j. antony; nischay mishra; sandra abel nielsen; ingrid lombardino, andrew cushing, emi knafo, rebecca eddy and danielle tarbert for clinical assistance, case submissions and case organization; ellie kahn for manuscript preparation; don schlafer, gerald duhamel the authors declare that they have no competing interests. key: cord- -b lesr g authors: clem, amy l; sims, jonathan; telang, sucheta; eaton, john w; chesney, jason title: virus detection and identification using random multiplex (rt)-pcr with '-locked random primers date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: b lesr g background: pcr-based detection and identification of viruses assumes a known, relatively stable genome. unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated pcr primer use problematic. furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to rt-pcr and dna chip detection. accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies. results: we report herein that viral genomic dna or rna can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single pcr using a mixture of primers designed to be resistant to primer-dimer amplification ( '-vvvvvvvvaa- ', v = a, g or c; ( )or primers). we have termed this novel pcr method random multiplex (rt)-pcr since hundreds of overlapping pcr amplifications occur simultaneously. using this method, we have successfullydetected and partially sequenced separate viruses in human plasma without using virus-specific reagents (i.e., adenovirus type , coxsackievirus a , and respiratory syncytial virus b). the method is sensitive to ~ genome equivalents/ml and may represent the fastest means of detection of unknown viruses. conclusion: these studies suggest that the further development of random multiplex (rt)-pcr may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology. relatively benign viruses can be converted into highly virulent viruses via the introduction of genes of interest. for example, ectromelia virus, a natural pathogen of mice that causes mousepox, recently was recombined with interleukin- as part of an effort to develop a live virus immuno-contraceptive vaccine. surprisingly, the recom-bined virus caused % mortality in strains of mice, whereas the wild type virus caused no death [ ] . a credible bioterrorism scenario might entail the release of such a recombined or chimeric virus tailored for maximum infectivity and pathogenicity but not readily detectable using our current "state-of-the-art" diagnostics (i.e., pcr and micro-array chips.) accordingly, there is a need for methods that can identify unknown viral pathogens and which can reveal extensive genomic information. such methods would not only prove useful for our defense against bioterrorism, but also would improve our capacities to identify and control outbreaks of naturally occurring pathogenic viruses. the proven technology to rapidly detect and identify known human pathogens as potential causes of disease or as bioweapons is pcr [ ] . a recent example involved a case of fatal yellow fever (yf) in a traveler returning from amazonas, brazil in march, [ ] . the otherwise healthy -year-old male developed fever, headache, pancytopenia, coagulopathy, renal and liver failure. pan-cultures were negative, and a peripheral smear yielded no plasmodia. serological tests (igg, igm) performed at the cdc were negative for yf, dengue, st. louis encephalitis, and other agents, but serum specimens (unfortunately examined only post-mortem) were positive for yf viral rna as measured by rt-pcr [ ] . subsequent attempts to isolate or culture the virus failed. this example highlights the superiority of pcr over other currently available methods. dna chips that allow the simultaneous measurement of literally thousands of genes through hybridization are now being developed as the next-generation rapid diagnostic test for all known human pathogens [ ] . however, both of these technologies rely on a relatively stable genome, and several human pathogens display a high mutation rate (e.g., hiv × - /base, kb genome [ ] ). moreover, the ability to recombine "non-pathogenic" viruses in vitro introduces the potential not only for de novo pathogenicity but also for enhanced stealth. these considerations suggest that it may be impossible to design dna micro-arrays which detect nucleic acids from all known and unknown viruses, including less obvious vehicles of bioterrorism such as adenovirus or rhinovirus recombined with a single gene for enhanced virulence. accordingly, there is a great need for the development of techniques that enable the universal amplification of viral nucleic acids. there are strategies to amplify genetic sequences with pcr without prior knowledge of the precise sequences. the first strategy relies on the degenerate binding of arbitrarily chosen primers to sample multiple cdnas or genomic dna species during pcr (two related methods are known as differential display and rna arbitrarily primed pcr [rap-pcr] [ ] [ ] [ ] ). the amplicons range in size from ~ - bp and overlap. rap-pcr was recently used successfully to identify a novel human pneumovirus only after the virus had been cultured [ ] . these techniques yield an amplicon "fingerprint" and are generally used to compare two populations of nucleic acids. accordingly, the pneumovirus was identified by comparing infected cells with non-infected cells [ ] . the other strategy is known as sequence-independent, single-primer amplification (sispa) and involves the directional ligation of a linker/adaptor oligonucleotide onto both ends of a target population of either ds dna or ds cdna (after reverse transcription from rna) [ ] . the pcr is accomplished with primers specific for the linked adaptor molecule. sispa, followed by extensive immunoscreening, enabled the cloning of the norwalk agent genome from stool and hepatitis g virus from plasma [ ] . unfortunately, these techniques have not found great utility in the direct discovery or identification of novel viruses from human plasma or serum samples (unless accompanied by immunoscreening) because of the relatively high level of host genomic dna. however, recent studies indicate that dnase treatment of serum samples can degrade most of the host genomic dna but not affect viral nucleic acids, which are protected by stable capsids [ ] . the combination of dnase treatment with sispa was successfully used to identify and sequence in their entirety novel parvoviruses from bovine serum [ ] . while the aforementioned pcr techniques are useful for the discovery of novel viruses, their utility to rapidly detect and identify unknown (or recombined) viruses is quite limited. rap-pcr, random rt-pcr and sispa are complicated multi-step procedures requiring infection of cultured cells, days to accomplish, expensive reagents and broad technical expertise. accordingly, these methods have not found clinical utility for the rapid detection or identification of viral agents. here, we present a novel method for rapid virus detection and identification based on random multiplex (rt)-pcr using '-locked random primers to avoid primer-dimer amplification. once detected, virus amplification products can be shot-gun cloned and sequenced for identification. this method may prove useful for the rapid detection and identification of emerging or recombined viruses. we hypothesized that random amplification of viral nucleic acids isolated from human plasma might provide a powerful new method to detect and identify novel and recombined viruses. allander et al. recently found that subjecting bovine plasma to filtration (to remove cells) and dnase treatment (to remove genomic dna) was sufficient to separate viral nucleic acids from bovine nucleic acids and thus enable sispa and cloning of novel viruses [ ] . the mechanism for this effect is related to the relative sensitivity of free-host dna to dnase treatment and to the relative insensitivity of virus capsid-protected dna to dnase treatment. we have found that human plasma is replete with free dna and rna species and thus opted to examine the potential of dnase and rnase treatment to isolate viral nucleic acids. we inoculated human plasma with adenovirus type and examined the effects of filtration, dnase treatment and rnase treatment on adenovirus type dna and on human β-actin genomic dna and mrna, using (rt)-pcr. in figure , we demonstrate that filtration, dnase and rnase treatment increases the efficiency of amplification of adenovirus type dna, whereas this combined treatment eliminates amplification of genomic β-actin (upper band) and β-actin cdna (lower band). although the addition of rnase alone has little effect on the β-actin mrna, the combination of dnase and rnase completely eliminates β-actin mrna. we postulate that this finding may be related to the protection of rna species by dna:rna hybrids. nevertheless, these data demonstrate that we are able to remove host β-actin dna and mrna without degrading adenovirus type dna. we next designed a mixture of random primers that would enable the priming and amplification of all dna or cdna molecules, but that would be resistant to primerdimer amplification. we chose base pairs as a compromise length to simultaneously optimize hybridization and possible binding sites. taq polymerase requires that the three ' bases of a primer be % complementary in order to enable efficient polymerization. we postulated that "locking" the ' end of the primers with adenosines and not incorporating any thymidines into the upstream ' portion of the primers would prevent primer-dimer amplification (i.e., '-vvvvvvvvaa- '; where v = a, g or c). this primer design will limit the frequency of binding sites within a particular viral genome, but using the edit-seq dna editing application (dnastar), we observed that primers within this mixture will bind every - bp in both orientations in the viral genomes that were examined, thus providing ample hybridization and amplification potential. as a first test of these random primers we employed dna plasmids of known sequence. we found that this mixture of primers enabled a continuum of discrete pcr products to be amplified from different circular dna plasmids ( figure a ). we shot-gun cloned these pcr products and confirmed that the amplified dna had derived from the source plasmids (data not shown). in contrast, primers consisting of the sequence '-nnnnnnnnnn- ' (where n = a, t, g, or c) did not enable the production of discrete or clonable pcr amplicons ( figure a ). we then examined the ability of the v aa primers to pcr amplify the plasmid, pbabe, and found that we could amplify as little as fg ( figure b ). we inoculated ml of human plasma with adenovirus type . the plasma was then filtered ( nm filter) and incubated for hours with nucleases. remaining nucleic acids were purified and pcr amplified using the '-vvvvvvvvaa- ' primers. we found that this mixture of primers enabled discrete pcr products to be amplified in the human plasma that had been inoculated with adenovirus type and treated with filtration, dnase and rnase treatment ( figure ). importantly, we observed only minimal amplification products in the un-inoculated plasma. we next shot-gun cloned the pcr amplicons into the topo . cloning vector and pcr screened colonies from e. coli that had been transformed with mixtures of recombined plasmids. in the un-inoculated plasma samples, we were unable to clone and sequence the amplicons (data not shown). in subsequent experi-filtration and nuclease treatment improves viral dna amplification by removing host dna and mrna figure filtration and nuclease treatment improves viral dna amplification by removing host dna and mrna. human plasma was inoculated with µl of an adenovirus type suspension, filtered and incubated for hrs with either u dnase or u rnase as indicated. remaining nucleic acids were purified with the qiaamp ultrassens virus kit and subjected to st strand cdna synthesis and cycles pcr using primers specific for either adenovirus type or human β-actin dna (upper band; primers cross an intron) or cdna (lower band). amplicons were then visualized on an ethidium bromide impregnated agarose gel. β-actin (genomic + cdna) + + + + + + + + filter dnase rnase + + + + + + + ments we have had variable success obtaining random multiplex pcr amplification from human plasma and have obtained only sequences in total from un-inoculated plasma. these sequences did not align with any known sequences in ncbi non-redundant database (blastn searches were conducted.) however, when we shot-gun cloned the dna amplified from the adenovirus -inoculated human plasma, we obtained recombined vector clones, and all clones matched to adenovirus type (see representative example, figure ). these data demonstrate that the combination of filtration, dnase/ rnase treatment and v aamer random multiplex pcr allows for the detection and identification of adenovirus type without using any virus-specific reagents such as primers, antibodies or microarray chips. the suspension of adenovirus type received from the atcc had not been quantified and the sensitivity of the random multiplex pcr method thus was uncertain. in order to generate a standard curve, we conducted realtime pcr of a known concentration of pcr-amplified adenovirus type genomic template using adenovirus type -specific primers and found that we could detect as little as copies/ml of template (data not shown). we then subjected the atcc adenovirus type suspension from the atcc to real-time pcr using the same adenovirus type -specific primers and found that we could readily detect the genomic dna (data not shown). based on the real-time pcr generated from the known concentrations of adenovirus type genomic template, we gen- g g a a g g t a a t a c a g c a c c g c t a t g c a c c c a c c c a g g a c c a c c c c t a a a a t c a a a t a : g c a a g g a a g g t a a t a c a g c a c c g c t a t g c a c c c a c c c a g g a c c a c c c c t a a a a t c a a a t a q u e r y : g g t t t t a g g a g t g t t a g t a t c a a a a a g a a g g t t a g g t g t t t c t g t a g c g g c t g t g c t g c t | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | s b j c t : g g t t t t a g g a g t g t t a g t a t c a a a a a g a a g g t t a g a t g t t t c t g t a g c g g c t g t g c t g c t q u e r y : g t c g g t a a c g t t c a c c a a a g t g a a a g t g t g g a a g c a a g g t c c g c t c t g g c a g t g g t a g g t | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | s b j c t : g t c g g t a a c g t t c a c c a a a g t g a a a g t g t g g a a g c a a g g t c c g c t c t g g c a g t g g t a g g t q u e r y : t c c c t c t a c a a a a g g a t t g t a g a g t a c t a g c t t a g t c t t t c t g g t a g t a t a a g t t a g t c c | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | s b j c t : t c c c t c t a c a a a a g g a t t g t a g a g t a c t a g c t t a g t c t t t c t g g t a g t a t a a g t t a g t c c q u e r y : a c t g g t a a g g t t g t t g g g t a t t t c a a t a c c g t c g t t g g c g c a g g c g t t g g a g a c t g c g a a c t : a c t g g t a a g g t t g t t g g g t a t t t c a a t a c c g t c g t t g g c g c a g g c g t t g g a g a c t g c g a a q u e r y : g g a a g t g t t t t t a a a a a g c c a g a g g a t g t a t t t g t c c c c c a g t c t g c a g t t g a a t t t t a c c t : g g a a g t g t t t t t a a a a a g c c a g a g g a t g t a t t t g t c c c c c g g t c t g c a g t t g a a t t t t a c q u e r y : c t c a g t c t g g t t g g t g a g g t t g a a g | | | | | | | | | | | | | | | | | | | | | | | | | s b j c t : c t c a g t c t g g t t g g t g a g g t t g a a g erated a standard curve plotting the concentration of adenovirus type genome equivalents versus the cycle number when fluorescence passed the pre-set threshold (ct; figure a ). based on this standard curve, we determined that the stock solution of adenovirus type contained × genome equivalents/ml. we then titrated down the concentration of virus in human plasma and conducted the combination of filtration, dnase/rnase treatment and random multiplex pcr using the '-vvvvvvvvaa- ' primers. we found that the random multiplex (rt)-pcr method is sensitive to genome equivalents/ml ( figure b ). we postulated that the random multiplex pcr method would also detect rna viruses if the viral nucleic acids were first subjected to st strand cdna synthesis. we inoculated human plasma with the rna virus coxsackievirus a (csv a ) and then filtered, treated with dnase and rnase, purified the remaining nucleic acids and conducted (rt)-pcr, using two commercially available reverse transcriptases (omniscript and superscript ii) and csv a specific primers separated by either bp or . kb. we found that whereas omniscript rt was more efficient at amplifying bp cdna species, superscript ii rt was more efficient at amplifying . kb fragments (figure a ). next, we used the '-vvvvvvvvaa- ' primers to random multiplex pcr amplify the cdna and observed distinct albeit faint amplicons ( figure b ). we cloned the amplification products and found that the amplified dna products matched the sequence of the cloned rna genomes as determined by blastn alignment searches of the ncbi non-redundant database ( figure ). we were surprised that the cloned dna from coxsackievirus a aligned with the known sequence for coxsackievirus a until we learned that the coxsackievirus a strain had not been fully sequenced and is thus not in the ncbi non-redundant database (search terms coxsackievirus and a ). accordingly, we had amplified portions of the coxsackievirus a genome that had not previously been sequenced (figure ) . we also inoculated human plasma with respiratory syncytial virus b and were able to amplify and identify genomic rna using the '-vvvvvvvvaa- ' primers (data not shown). however, the pcr amplification efficiency was poor in both rna viruses relative to the dna virus, av , and we suspect that this may be related to the efficiency of the reverse transcriptases or the stability of the rna. in november , an outbreak of atypical pneumonia occurred in guangdong province, people's republic of china [ , ] . the disease did not respond to empirical antibiotic treatment, and no known bacterial or viral pathogens were identified using serological and (rt)-pcr analyses. the disorder was called severe acute respiratory syndrome (sars), and by july it had caused reported cases and fatalities [ , ] . in an effort to identify the causative agent of sars, the who sars aetiology study group coordinated the distribution of information and infected materials and the analyses of the data real-time pcr quantitation of adenovirus type dna template figure real-time pcr quantitation of adenovirus type dna template. a. pcr-amplified genomic dna from adenovirus type was quantitated by spectrophotometry (od nm) and the genome equivalents/ml calculated based on avogadro's number ( . × /mol). realtime pcr was conducted with adenovirus type specific primers using to genome equivalents/ml as template and then using µl of the stock solution of adenovirus type as template. a standard curve was produced based on the threshold cycle number of the tested concentrations of adenovirus type dna template and plotted compared to the threshold cycle number (c t ) of the stock solution of adenovirus type . b. the indicated genome equivalents of adenovirus type were added to ml of human plasma, treated with nm filtration and dnase/rnase digestion. total nucleic acids then were purified, amplified by random multiplex pcr ( cycles) and sequenced as described in the methods section. cycle number (ct) amongst eleven separate laboratories [ ] . researchers cultured fetal rhesus kidney cells with infected nasopharyngeal aspirates and then conducted random rt-pcr using the primer, '-gccggagctctgcagaattc-nnnnnn- ' for reverse transcription, and '-gccg-gagctctgcagaattc- ' for pcr. unique pcr products in the infected cell preparation were cloned and sequenced, and % homology was found with bovine coronavirus [ ] . rt-pcr of hundreds of infected clinical specimens, using primers specific for the novel virus, con-firmed the identification [ , ] . while this approach was successful, it is worthwhile to note that it required months and laboratories to identify the virus. by contrast, the procedures described herein are -at least theoretically -capable of identifying an unknown virus without the use of specially designed primers. furthermore, is possible to at least partially identify an unknown agent within - days in one laboratory without intermediate culture steps. in the current study, we have detected and identified separate viruses (i.e., adenovirus type , coxsackievirus a , respiratory syncytial virus b), using the novel approach of random multiplex (rt)-pcr of purified viral nucleic acids. the detection was accomplished in hours using only a micro-centrifuge and pcr machine and did not require any virus-specific reagents. the sensitivity is genome equivalents/ml for adenovirus type which is not as sensitive as virus-specific pcr (< genome equivalents/ml) but should be adequate for the detection of free virus in untreated individuals. coupled with the potential for direct sequence information, we are confident that this approach may prove useful for our defense against acts of bioterrorism, as well as for the detection and characterization of novel viruses in blood products and in patients displaying hallmarks of infection (e.g., leukocytosis, fever). although we believe that the method is adequate in current form to test clinical specimens in a randomized, blinded diagnostic trial, we consider that certain alternative approaches may yield a method that has improved sensitivity and specificity. in particular, we intend to decrease the background amplification that has been variably observed in order to prevent false positives and to increase sensitivity by applying sybr green fluorescence for dna detection. finally, we will apply the optimized approach to blood specimens from virally infected patients in order to detect unsuspected variables and to validate the method within the clinical environment. our long-term goal is to provide emergency room and primary care physicians with the ability to universally detect viruses in human plasma. the rationale is, in part, that if an emerging or recombined pathogenic virus were intentionally released into a population, then the ability of physicians to detect the pathogen will be crucial for containment. a second and related application of this technique involves identification of previously undescribedbut naturally occurring -viruses in patients suffering with idiopathic disease states of possible viral etiology. a third application of these procedures may be in the screening of blood bank blood just for the presence of virus. although the precise identification of the virus requires cloning and sequencing, the amplification pattern will provide an most clinical laboratories that service emergency rooms and primary care clinics possess the ability to conduct rt-pcr for known viruses such as hiv, and the unmodified random multiplex (rt)-pcr method does not require any further technical expertise. in conclusion, the optimization and testing of the random multiplex (rt)-pcr technology should allow for the development of a universal virus detection assay that will vastly improve our collective defense against viral bioweapons and against emerging, heretofore unidentified natural viruses. plasmids ( isolation of virus from plasma ml of blood plasma ± virus was cooled to room temperature and placed into a ml tube. viral isolation was carried out according to the manufacturer's protocol (qiagen). viral dna or rna was eluted off the column in a final volume of µl elution buffer. this product was either used directly for random pcr or used in the reverse transcription reaction. reverse transcription was carried out according to the manufacturer's protocol either with the omniscript rt (qiagen) or superscript ii rt (invitrogen). the maximum amount of viral rna was used in place of water for both reactions. the pcr of viral dna isolated from whole blood was done according to the method described above utilizing the '-vvvvvvvvaa- ' primers. products were run on a % agarose gel containing ethidium bromide. the bands which differed from the water control were excised and the dna was extracted using the qiaquick gel extraction kit (qiagen). dna was eluted off in a final volume of µl elution buffer. the dna was cloned into the topo cloning vector (invitrogen) following the manufacturer's protocol and transformation was carried out in top chemically competent e. coli. resulting colonies were picked randomly and mixed with µl pbs. µl of this mixture was used in the pcr reaction using the universal m forward and reverse ( µl of µm) primers and × pcr master mix (promega). the following parameters were used for colony screening: °c- minutes, and then cycles of °c- seconds, °c- seconds, °c- minute. pcr products were run out on a % agarose gel containing ethidium bromide. those colonies which yielded products greater than bp were grown in lb broth overnight and the dna was isolated according to the manufacturer's protocol (qiagen) eluting the dna off in a final volume of µl. dna was sequenced using the universal m forward and reverse primers at the university of louisville's core sequencing facility. resulting sequences were screened using the blast program on the ncbi web site to determine sequence origin. pcr was carried out with either viral dna or cdna using a × pcr master mix (promega) and µl of viral dna. µl of µm primers was used for each reaction. the total volume was equal to µl per reaction. the following parameters were used for viral-specific amplification: °c- minutes, °c- seconds, °c- seconds, °c- minutes, repeat times, °c- minutes. generating viral dna templates for real time pcr viral dna isolated from whole blood plasma was used to generate a template for use in real time pcr. µl of dna from the virus was used with platinum pcr master mix (invitrogen) and µl of a µm solution of each of the template primers outlined above. the total volume of the reaction was µl. the same pcr parameters were used as were used for viral-specific amplification. the resulting products were run on a % agarose gel containing ethidium bromide. the bands were excised and dna was eluted according to manufacturer's instructions (qiagen). all real time pcr samples were run on a cepheid smart cycler. omnimix hs reagent (cephied, packged by takara) was re-suspended in µl of water, which was sufficient for two pcr reactions. µl of the stock virus and µl of each of the template primers ( µm) were added and mixed. samples were run at °c for sec, °c for sec, and °c for sec for a total of cycles. the molecular weight was determined for each of the dsdna templates generated by pcr. the od of the resultant dna was also recorded. the od, in grams/µl was divided by the molecular weight of the product and the result was equal to moles/µl. this number was then multiplied by avogadro's number ( . × molecules/ mole) and the resulting number is equal to the number of viral dna molecules per microliter. expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox detection of smallpox virus dna by lightcycler pcr fatal yellow fever in a traveler returning from amazonas, brazil sequence-specific identification of pathogenic microorganisms using microarray technology estimation of effective population size of hiv- within a host: a pseudomaximum-likelihood approach differential display of eukaryotic messenger rna by means of the polymerase chain reaction a novel usage of random primers for multiplex rt-pcr detection of virus and viroid in aphids, leaves, and tubers rna fingerprinting using arbitrarily primed pcr identifies differentially regulated rnas in mink lung (mv lu) cells growth arrested by transforming growth factor beta osterhaus ad: a newly discovered human pneumovirus isolated from young children with respiratory tract disease sequence-independent, single-primer amplification (sispa) of complex dna populations the isolation and characterization of a norwalk virus-specific cdna a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species osterhaus ad: newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome this study was funded by a grant from the center for genetics and molecular medicine, university of louisville. the author(s) declare that they have no competing interests. key: cord- - zyv h authors: jonsdottir, hulda r.; dijkman, ronald title: coronaviruses and the human airway: a universal system for virus-host interaction studies date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: zyv h human coronaviruses (hcovs) are large rna viruses that infect the human respiratory tract. the emergence of both severe acute respiratory syndrome and middle east respiratory syndrome covs as well as the yearly circulation of four common covs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. various animal models have been established to investigate hcov infection, including mice and non-human primates. to establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. currently, different cell culture systems are available to recapitulate the human airways, including the air-liquid interface (ali) human airway epithelium (hae) model. tracheobronchial hae cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. these organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. no commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. these morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition. respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. currently, they are studied in various model systems, ranging from cell lines to animal models. originally, the importance of hcovs in the burden of human disease was underestimated and as a result, no general therapy exists to treat coronavirus induced disease in humans. furthermore, no commercial vaccine is available leaving the human population vulnerable to emerging coronavirus infections. both the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses have recently crossed the species barrier and entered the human population to cause severe disease. in this review, we summarize the current knowledge on human coronavirus infection emphasizing the usefulness of organotypic human airway cultures as a model system. coronaviruses (covs), a subfamily of the coronaviridae family, are positive strand rna viruses with the largest genome of all known rna viruses (≥ kb). the genomic rna is capped, polyadenylated and associated with nucleocapsid proteins within an enveloped virion. the envelope is covered by the characteristic surface glycoprotein that gives the virus particles their characteristic crown-like (latin: corona) appearance [ ] . all covs share a common genome organization where the replicase gene encompasses the ′-two thirds of the genome and is comprised of two overlapping open reading frames (orfs), orf a and orf b that encode for up to non-structural proteins. the structural gene region, which covers the ′-third of the genome, encodes the canonical set of structural protein genes in the order ′ -spike (s) -envelope (e) -membrane (m) and nucleocapsid (n) - ′. the structural gene region also harbors several orfs that are interspersed along the structural protein coding genes. the number and location of these accessory orfs vary between the cov species [ , ] . in animals, cov infections are mainly associated with respiratory and enteric disease and can have large economical impact on the veterinary industry, e.g. porcine epidemic diarrhea virus (pedv) causes gastrointestinal disease in pigs [ ] , infectious bronchitis virus (ibv) causes severe kidney and respiratory disease in chicken [ ] and bovine coronavirus (bcov) causes both respiratory disease and diarrhea in cattle [ ] . additionally, cov infections can have other disease manifestations, such as central nervous system (cns) involvement, hepatitis and peritonitis [ ] [ ] [ ] [ ] . in humans, cov infections are mainly associated with respiratory diseases that are considered to have a large impact on the economy due to reduced productivity of the working population. currently, coronaviruses that cause disease in humans have been discovered. four of those are commonly circulating and two have caused epidemics of severe acute respiratory disease. the first human coronavirus (hcov), b , was described in . in the following years, over additional strains were characterized. ten of those strains could only be isolated from primary embryonic tracheal organ culture. others were readily isolated from monolayer cultures and were antigenically related to the prototype strain hcov- e. hcov-oc , for organ culture , was isolated and found to be distinct from the e prototype strain [ , ] . in the subsequent decades, research on hcovs would center on these two distinct viruses. however, in , an unknown respiratory illness, termed severe acute respiratory syndrome (sars), surfaced in asia. research determined it to be caused by a novel coronavirus [ , ] . at the end of the epidemic, this virus had infected over people, most in china, and caused deaths [ ] . following the discovery of this virus, two additional covs causing human disease were identified. hcov-nl was isolated in the netherlands in from an infant with bronchiolitis [ ] and hcov-hku in from a patient with pneumonia in hong kong [ ] . in , another respiratory hcov, middle east respiratory (mers)-cov, was isolated from a patient with pneumonia in saudi-arabia [ ] . unlike sars-cov, this virus is still intermittently present in the human population and most recently caused a large outbreak in south-korea [ ] . to date, there have been over cases and almost deaths related to mers-cov infection [ ] . out of the known human coronaviruses, hcov- e, hcov-oc , hcov-nl and hcov-hku are commonly circulating in the human population and usually cause general respiratory illness and cold symptoms in healthy individuals [ ] [ ] [ ] . like influenza, these viruses are capable of causing more severe disease in the immunocompromised and the elderly [ ] . they infect the human airway from the luminal side and progeny viruses are released from the same side facilitating spread through coughing and sneezing [ , ] . these coronaviruses are responsible for approximately - % of all upper and lower respiratory tract infections [ ] [ ] [ ] but the interactions between them and their natural host cells are poorly understood. currently, it is hypothesized that most of the human coronaviruses may have originated from bats [ , ] . for example, hcov- e is believed to originate from african hipposiderid bats possibly using camelids as intermediate hosts [ ] . in the last years, two coronaviruses have crossed the species barrier and caused severe and fatal disease in humans. sars-cov surfaced in and mers-cov in [ , , ] . as opposed to the commonly circulating viruses, which generally only cause mild respiratory symptoms, these viruses presented with higher case fatality ratios, around and - % respectively [ , ] . currently, there is abundant phylogenetic evidence for the bat origin of sars-cov, based on sequences of sars-like viruses found among bats in the recent years [ ] [ ] [ ] . the initial transmissions of sars-cov from animals to humans were traced back to the live animal wet markets and it was hypothesized that the virus made its way into the human population using the civet cat as an intermediate host. however, successful isolation of sars-like viruses from bats [ ] and the fact that a contemporary bat sars-like virus can infect human airway cultures [ ] suggest that an intermediate host between humans and bat might not have been needed for the transmission of sars-cov. the evolutionary origin of mers-cov is less clear but it has been speculated to be bats as well. characterization of an african bat virus closely related to mers-cov shows that both the human and camel strains belong to the same viral species and phylogenetic analysis suggests that mers-cov infection in camels predates that in humans, suggesting that camels infect humans and not the other way around. furthermore, the bat virus roots the phylogenetic tree providing further evidence for the bat origin of mers-cov [ ] . additionally, human-tohuman transmission, although not robust, seems to happen simultaneously as camel-to-human transmission. therefore, any further adaptation of mers-cov to the human host must be monitored carefully and intermediate hosts identified [ ] . many bat coronaviruses have been identified in the recent years further highlighting the zoonotic potential of this family of viruses [ ] . given the documented history of coronaviruses overcoming the species barrier and causing severe disease in humans, it is important to investigate the zoonotic potential of close evolutionary relatives of common hcovs in a culture model that recapitulates the aspects of the human airway, e.g. morphology and receptor distribution. it's important to study the mechanisms of pathogenesis and the evolution of zoonotic viruses in detail in order to identify molecular determinants that affect either transmission or pathogenesis. it's also important to elucidate whether coronaviruses currently circulating in animals are a potential danger to the human population. all of the known cellular receptors of hcovs belong to the same protein family, the membrane ectopeptidases. interestingly, the catalytic activity of these peptidases is not required for viral entry but rather the co-expression of other host peptidases activates the hcov spike proteins [ , ] . it has been established that the human transmembrane serine proteases tmprssii and hat cleave and activate the hcov- e, sars-and mers-cov spike proteins during viral entry [ , ] . out of the four commonly circulating coronaviruses, hcov- e is the only one that infects non-ciliated cells using the human aminopeptidase n (hapn) as its receptor [ ] . this peptidase is predominantly expressed on non-ciliated cells in the human bronchus [ ] . sars-cov and hcov-nl both utilize the angiotensin converting enzyme (ace ) for cellular binding [ , ] . ace is expressed on ciliated bronchial cells along with endothelial cells and both type i and ii alveolar cells [ ] . mers-cov was found to use a different receptor than sars-cov, namely the dipeptyl-peptidase (dpp ) [ ] . dpp is widely expressed in endothelial cells and various epithelial tissues in the human body [ ] . in ex vivo human lung organ cultures, different tropism of sars-and mers-covs was observed. mers-cov can actively replicate in both bronchial and alveolar tissue while sars-cov primarily replicates in alveolar tissue [ ] . the wide cellular tropism of mers-cov might contribute to its associated disease severity and high mortality rate whereas the alveolar replication of sars-cov would explain why it generally presents with pneumonia. the cellular surface receptors for hcov-oc and hcov-hku are currently unknown but receptor determinants for these two viruses have been identified as n-acetyl- -o-acetylneuraminic acid and o-acetylated sialic acid, respectively [ , ] . all of these viruses can be successfully cultured and investigated in hae cultures [ , ] . the discovery of hcovs, their receptor usage, cell tropism and receptor binding domain (rbd) is summarized in table . furthermore, established reverse genetic systems for hcov- e [ ] , hcov-oc [ ] and hcov-nl [ ] allow for controlled virus mutation and fluorescent transgene insertion to better understand the interaction of these viruses with their pulmonary host cells. traditionally, respiratory viruses are studied in animal models, usually mice and ferrets [ , ] . however, it is not always possible to correctly recapitulate human infection and disease in animal models. the establishment of transgenic animal models for human disease is attainable when either the virus receptor has been identified, which is not the case for all hcovs, or when viruses can be adapted to a different host. an adapted human virus may not share the same properties as the original human virus. sars-cov was found to replicate naturally in various strains of inbred mice but to enhance clinical signs of disease the hace was introduced into these mice. this resulted in murine models with varying degree of human disease similarity. since sars-cov already replicated in mouse cells, adapting it to the murine host was quite successful. this resulted in three mouse adapted strains that caused disease in mice similar to severe sars-cov cases in humans [ ] . in an effort to establish a mouse model for hcov- e infection transgenic hapn mice were created. however, the insertion of the hapn into mouse cells is not enough to establish robust hcov- e infection in vivo. nevertheless, cells isolated from these transgenic animals could be infected in vitro [ , ] . the emergence of both sars-and mers-covs emphasized the importance of establishing animal models for human coronaviruses. currently, a few animal models for mers-cov have been established. mice carry their own variant of the viral receptor ddp that differs from the human in regions important for mers-cov spike interaction and by replacing this receptor with the human one, mers-cov can infect mouse cells but the method of hdpp insertion has an effect on the degree of pathogenesis observed in these mice [ , ] . various non-human primates (nhps) can be naturally infected with both sars-and mers-covs. however, disease presentation and pathogenesis differs between the different subspecies and nhp models are expensive, although ideal to study human infection due to their genetic similarity [ ] . to establish a link between the research conducted in animal models and humans, an organotypic airway culture system resembling the human airway epithelium has been developed. this model is a universal platform to study human respiratory viruses [ ] [ ] [ ] [ ] . they have been used successfully for infection studies with all known human coronaviruses [ , ] . furthermore, the cultures can be inoculated with a low infectious dosage to mimic natural infection in the human airway. whereas, animal models often require both high dosage and artificial inoculation routes. organotypic cell cultures are becoming increasingly common. different cell culture models exist to depict different epithelial tissues [ ] . these cultures closely resemble their tissue of origin and contain various different cell types with distinctive roles in the polarized tissue. currently, various organotypic cell culture models exist to represent the different areas of the human airways. the human lungs span a long anatomical distance and carry out different functions depending on anatomical location [ , ] . the structure of the epithelium also differs the further you descend into the airways. tracheal and bronchial epithelium is columnar and pseudostratified, with every cell in contact with the basement membrane, while the epithelium in the alveoli is comprised of a single cell layer to facilitate air-exchange [ ] . tracheobronchial cells are one of the first targets of human respiratory viruses and can be cultured in airliquid interface (ali) where the apical side of the cell layer is exposed to air while the basolateral side is submerged in medium. tracheobronchial cells cultured in that way form a pseudostratified epithelial layer that both morphologically and functionally resembles the human upper conducting airway (fig. a) [ , ] . after differentiation, these cultures contain many different cell types such as basal, ciliated and goblet cells. they also produce protective mucus, much like in vivo epithelium. when compared to primary bronchial cells in submerged two-dimensional culture, the gene expression of primary ali cultures differs significantly. however, the expression pattern of primary human bronchial ali cultures is comparable to that of in vivo epithelium. the human bronchial cell line calu- has been used as a culture model for respiratory epithelium but its gene expression in ali cultures is more similar to submerged bronchial cell cultures than differentiated epithelium [ ] . disruption of the cell layer [ ] . therefore, the primary tracheobronchial ali culture model is especially fitting for human respiratory virus research since it accurately recapitulates the primary entry point for these viruses. by using these cultures, virus replication and host interactions can be studied in natural target cells. further establishing the usefulness of this system hcov-hku was propagated for the first time in ciliated cells of bronchial hae cultures in after culturing it in conventional cell lines had failed [ ] . alveolar epithelial ali cultures (fig. b) can also be used for virus-host interaction studies and are especially applicable when a viral infection causes pneumonia and alveolar damage [ ] . hcov-hku has also been propagated in alveolar hae cultures and exhibits a strong tropism for alveolar type ii cells and causes large syncytia formation upon infection [ ] . when compared to traditional two dimensional cell cultures, the hae cultures are more cumbersome and their preparation is time consuming but they do have an advantage over traditional monolayer cell cultures when it comes to virus-host interaction studies. different types of ali cultures used for virus research are summarized in table . within the respiratory epithelium the innate immune system has a major protective role as the first line of defense against respiratory pathogens. in particular, the interferon (ifn) system orchestrates hundreds of different cellular effector proteins that (i) protect the epithelial barrier by altering the physiological and cellular environment, (ii) impair virus propagation, spread and transmission, and (iii) shape the host's adaptive immune response. recent publications have demonstrated that the innate immune system is functional in the hae cell culture system and that most pathogen recognition receptors are expressed and up-regulated upon treatment with exogenous stimuli [ , ] . in general, hcovs do not elicit a strong innate immune response in primary target cells of the human airway early during infection. despite the presence of all major pathogen recognition receptors, no elevated expression of ifn beta, pro-inflammatory cytokines or interferon stimulated genes can be observed up to h post-infection in haes infected with hcov- e, mers-or sars-covs [ ] . this is most likely due to the intrinsic cov properties harbored in the replicative non-structural proteins that actively aid in avoiding recognition by the host innate immune system. for example, the ′ termini of the viral mrna are capped making them indistinguishable from the host cellular mrnas and no longer detectable by cellular sensors. furthermore, cov replication is associated with the appearance of double membrane vesicles (dmvs) in the host cell cytoplasm, which might serve as a protective shield for viral rna to prevent recognition by cytoplasmic rna sensors [ ] [ ] [ ] [ ] . in addition to the non-structural proteins, various cov accessory proteins have been discovered to inhibit interferon signaling at different stages of the host innate immune response. for example, mers-cov accessory protein a inhibits innate antiviral signaling by suppressing the activation of mda and rigi [ , ] whereas b inhibits the induction of the ifn-beta promoter [ ] . while orf a and b are ifn antagonists in the genome of mers-cov, sars-cov orf b antagonizes ifn signaling through mavs/rigi [ ] . whereas sars-cov orf disrupts ifn signaling by blocking the nuclear translocation of stat [ , ] . these discoveries highlight that hcovs employ similar yet different strategies despite that respiratory infections with hcovs can result in severe respiratory disease there are currently no effective prophylactic or therapeutic treatment options available. however, the emergence of novel coronaviruses has emphasized the need to develop effective treatment options. for example, vaccines using the spike proteins of both sars-and mers-covs have proven protective in animal models [ , ] suggesting that a vaccine against hcovs for human use might be achievable. additionally, various drugs that inhibit hcov infection at different stages of the replication cycle have been reported and some could potentially serve as treatment options for hcov associated severe respiratory disease. for example, patients presenting with severe respiratory disease, caused by sars-or mers-covs, are generally treated with steroids and interferon, sometimes in combination with the antiviral drug ribavirin [ ] [ ] [ ] [ ] . this treatment, however, is not especially effective highlighting the need for hcov specific antivirals. many different compounds have been determined to have anti-hcov activity. for example, protease inhibitors which suppress hcov entry [ ] [ ] [ ] , cyclosporin a (csa) treatment blocks the replication of coronaviruses from all subgroups [ ] and non-immunosuppressive derivatives of csa represent a possible therapeutic option for both human and animal cov infections. hcov infection can also be inhibited by pre-treating hae cultures with either recombinant ifn alpha or lambda [ ] . similar effect has also been shown for recombinant ifn alpha and beta which could inhibit mers-cov in ex vivo lung cultures [ ] . as previously described, ifn treatment of active hcov infection is not particularly effective in vivo. therefore, the use of ifn in humans might be limited to prophylactic treatment of exposed persons and/or health care workers treating infected patients. screenings of compound libraries have also resulted in the identification of some hcov specific antivirals. for example, a novel small compound inhibitor (k ) has been identified, and showed to be effective against a broad spectrum of covs and could inhibit both hcov- e and mers-cov in hae cultures [ ] . additionally, hcov-nl has been inhibited in hae cultures with polymer-based compounds [ ] . to date, most treatment and inhibitor studies have been conducted in hcov susceptible cell lines. however, the hae cultures represent an ideal system to test the application and efficacy of those already identified, and new, antiviral compounds against hcovs in cells that represent the primary site of replication. furthermore, the hae cultures are heterogenous, containing many different cellular sub-populations, and would allow for the evaluation of compound toxicity and effect in a differentiated layer similar to human airway epithelium. compounds already shown to inhibit hcovs in cell lines should be applied to hae cultures as well before any animal or human trials. hcov induced respiratory diseases are of both medical and socio-economic importance. the emergence of sars-and mers-cov and the yearly circulation of the four common hcovs highlight the importance of elucidating the different mechanisms employed by hcovs to evade the host immune system as well as identifying antiviral compounds and human vaccine candidates. the hae culture system is based on primary human cells offering a unique platform to study respiratory viruses in cells representing the primary entry point of these viruses, bronchial epithelial cells, or investigate the interaction of hcovs and the distal airways, in type i and ii alveolar cells. additionally, the inclusion of airway epithelial cultures for other species enables the study of zoonosis and animal-to-human transmission. currently, many aspects of hcov infection and pathogenesis remain to be determined. the hae culture system, both tracheobronchial and alveolar, represents a unique platform to study virus-host interaction in natural target cells at the molecular level. these cultures are becoming more common and more relevant to hcov research. especially, for those viruses for which there is no animal model, as they provide an organotypic substitute for virushost interaction studies. the authors declare no competing interests. authors' contribution hrj wrote the review, designed tables and figures. rd revised the text, tables and figures. both authors read and approved the final manuscript. this work was supported by the r foundation, switzerland (project - ) and the university of bern initiator grant. received: december accepted: january coronaviruses: an overview of their replication and pathogenesis coronavirus genome structure and replication the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses a new coronavirus-like particle associated with diarrhea in swine a nomenclature for avian coronavirus isolates and the question of species status enteric disease in postweaned beef calves associated with bovine coronavirus clade cleavage of a neuroinvasive human respiratory virus spike glycoprotein by proprotein convertases modulates 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sars coronavirus dipeptidyl peptidase distribution in the human respiratory tract: implications for the middle east respiratory syndrome we would like to thank prof. dr. volker thiel for his careful review of the manuscript and dr. eveline kindler for providing components for figures. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -qc pm authors: ji, jun; xie, jingwei; chen, feng; shu, dingming; zuo, kejing; xue, chunyi; qin, jianping; li, hongmei; bi, yingzuo; ma, jingyun; xie, qingmei title: phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during - date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qc pm background: the nephropathogenic avian infectious bronchitis (ib) caused unprecedented economic losses to the commercial chicken industry of china in - . to investigate the prevalence of nephropathogenic ib in china, eighty ibv isolates from different provinces during - were identified by dwarf embryo test and rt-pcr. results: the strains were mostly isolated in winter and spring with a wide age range of ib outbreaks, from to days. by the virus recovery trials, / of the strains resulted in the deaths or distresses of birds from nephritis. to learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike (s ) protein gene of these strains was rt-pcr amplified and sequenced. compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the s genes of these strains and the reference strains displayed homologies ranging from . % to . % and from . % to . % respectively. s protein of the major pandemic strains contained or amino acids with the cleavage site of hrrrr or rrfrr. phylogenetic analysis revealed that recent field isolates of ibv in china were mostly belonged to a -branch (qxibv-branch) and hn -branch, only one isolate was belonged to gray-branch and m -branch respectively. most of the strains showed evolutionarily distant from vaccine strains. conclusions: the results of this study suggested that nephropathogenic ibvs were mainly a -like strains in china during - . infectious bronchitis (ib) is a serious and highly contagious disease of chickens, accompanied by decreased egg production and poor egg quality in laying flocks. avian infectious bronchitis virus (ibv) was first reported in the usa, replicating in the respiratory tract and some epithelial cells of gut, kidney, and oviduct [ ] [ ] [ ] . ibv commonly predisposed the birds to secondary infection with some bacterium, such as escherichia coli and mycoplasma gallisepticum, resulting in complicated disease process and increased mortality [ , ] . the clinical disease and production problems frequently cause catastrophic economic losses to the poultry industry all over the world. ibv belongs to the genus coronaviridae, family coronaviridae, order nidovirales, and possesses a single stranded positive-sense rna genome encoding four structure proteins, phosphorylated nucleocapsid (n) protein, small envelope protein (e), integral membrane glycoprotein (m), and spike glycoprotein (s) [ , ] . the s glycoprotein on the outside of the virus contains epitopes associated with serotype differences, and is cleaved post-translationally by cellular proteases into the s and s subunits [ , ] . the globular s subunit forms the tip of a spike, extending outward, plays a role in attachment and entry into the host cell, which has relation to induce virus neutralizing antibody and hemagglutination inhibition antibody, whereas the s subunit anchors the s moiety to the viral membrane [ ] [ ] [ ] [ ] . coding for the heavily glycosylated spike glycoprotein, the error-prone nature of rna polymerase made the s gene could easily generate nucleotide insertions, deletions, point mutations, and rna recombination under vaccine pressure, to bring about new variation strain and change of tissue tropism [ ] [ ] [ ] [ ] [ ] . it is documented that only a few amino acid differences amongst s proteins are sufficient to have a detrimental impact on cross-protection [ , [ ] [ ] [ ] [ ] . antigenically different serotypes and newly emerged variants of field chicken flocks lead to vaccine breaks [ , ] . recently, more than serotypes within ibv have been identified worldwide. the complex epidemiology characterize of ib raised the control difficulty. in china, since ibv strains were first isolated and identified in , various live-attenuated and inactivated vaccines derived from massachusetts (mass) serotype strains have been widely and extensively used in chicken farms to reduce the adverse effect of the ibv [ , ] . however, the disease continues to emerge and cause serious production problems, even occurred in routinely vaccinated layer and breeder flocks in china, and the situation gets worse as time progressed [ ] . it was documented that nephropathogenic type ib has become more and more prevalent in china. the unprecedented economic losses caused by the nephropathogenic ib suggested that selecting the appropriate vaccine strain against the ib outbreaks is of great importance [ , ] . however, the integrated natures of novel circulating ibv strains in mainland china were not well-learned. the previous study by other researchers has been revealed that the variation in s sequences was closely confirmed relative to the emergence of novel strains, and s gene sequence was a good predictor of challenge of immunity in chickens [ , , ] . this study was conducted to identify the ibv strains that have escaped immune defenses conferred by vaccination in china. the genetic characterization of recent ibv field isolates in china was performed by sequencing the whole s genes, sequence alignment and phylogenetic analysis compared with other reference strains. from unhealthy birds suspected of ibv infection in the vaccinated chicken flocks from guangdong, guangxi, fujian, hainan, jiangsu, zhejiang, chongqing, hubei, sichuan and jiangxi province of china, filed ibv strains were isolated during - . the isolation rates in the two years were season-dependent to some extent, strains were isolated in october, while only seven strains were isolated in summer (from june to august). the ages of flocks at the time of the outbreak varied between and days. most of the strains were isolated from the chickens between to days of age. the detailed clinical record of each strain was showed in table . after three passage propagation, ibvs of all isolates induced peripheric lesions and growth retardation of embryo at h post-inoculation. since the fourth day post-inoculation, most of the chicks were listless and huddled together, showed ruffled feathers. the results of virus recovery in chicks indicated . % ( / ) isolates caused serious kidney lesions, which were presented with swollen specked kidney and distended ureters filled with uric acid were nephropathogenic type, and the other ten isolates in the study caused respiratory system signs, which were consistent with the clinical record of each strain (table ) . homologies among s nucleotide and deduced amino acid sequences the obtained strains were characterized phylogenetically by nucleotide sequence analysis of the hyper-variable s gene of ibv. the nucleotide and amino acid sequence similarities between the eighty ib strains were ranging from . % (strain cq and hy) to % (strain pt and pt ) and . % to %, respectively. compared to the reference strains published in the genbank, the identity of the nucleotide and amino acid sequence among the isolates (including the isolates in this study plus the reference strains) were . to . % and . to . %, respectively, indicating low homology and high variation among the isolated and reference strains. as shown in the table and table , s genes of the newly strains contain mutations, insertions and deletions, resulting in different lengths of nucleotides. s genes of these strains were generated and confirmed from three time sequencing results, contained , , , , , and nucleotides, amino acids sequences ranging from (lc strain) to (lc strain). the length differences indicated amino acid insertions and deletions exist among the different strains. through the alignment analysis, the deletions, insertions and mutations of each obtained s gene was summarized in table . most variations in the deduced amino acid sequences of chinese ibvs were observed among residues - , - and - (numbering was with reference to s sequence of the mass strain). the precursor protein of s glycoprotein is cleaved into amino-terminal s and s protein by the protease during viral maturation [ ] . in this study, the most common cleavage recognition sites of s gene were rrf(s/l) rr ( / ) or hrrrr ( / ) in the china field strains ( table ). the exceptional ones included cq (rrtgr), hy (rrskr), and hy (rrskr). the cleavage sites of these two strains containing amino acids k, t, and g, were novel motifs compared to the reference strains, and quite different with the other isolates of the cleavage site. a phylogenetic tree was constructed from the nucleotides sequences of the s glycoprotein genes. as shown in the figure , the isolates ibv strains were clustered into five distinct genetic groups or genotypes which were considerably heterogeneous, including a type ( newly isolated strains), / -type ( newly isolated strains), hn -type ( newly isolated strains), gray-type and m -type. the newly isolated strains mainly belonged to a -type, / -type and hn -type branch. the phylogenetic relationship of strains at different times and geographical regions displayed complexity and diversity. strains isolated from hubei, zhejiang, jiangsu, guangdong, guangxi and fujian province mainly belonged to the a branch, also including other seven published ibv strains from china (qxibv, ck/ch/ljl/ ii, ck/ch/ ljs/ iv, ck/ch/lsd/ - , ibvsx , lz and lz ). the isolated strains of hainan province and a few isolated strains from guangdong and fujian province belonged to the hn branch, included psh and ck/ch/lcq/ ii. group gray-type was correlative with the american strain (gray), included other two classical american strains (ark and holte), one japanese strain (jp ), and the exceptional field strain (cq ). most of the current vaccine strains (h , h , ma , m , w , / and / ) were belonged to the m branch, which including one field strains (nj). however, the current pandemic strains were mostly / -type, a -type (qxibv-type) and hn -type, indicating that the field ibvs co-circulating in chicken flocks in china were evolutionarily distant from the known vaccine strains. infectious bronchitis (ib) is one of the most common and difficult-control poultry diseases in china, caused persistent but infrequent outbreaks in commercial chicken farms [ , , ] . commercial vaccines based on h , h , / , ma , w and m strains, have been widely used to control the disease [ , ] . natural outbreaks of ibv often are the result of infections with strains that differ serologically from the vaccine strains. come to the rapid and complicated evolutionary of ibv, it is imperative to learn profoundly the circulating ibvs, facilitate selecting the candidate vaccine strain against the infections [ , ] . in this study, ibv strains were isolated from the vaccinated chicken flocks, with a wide age range of ib outbreak. the chickens infected before the age of days which might be caused by the vertical transmission of ibvs or the maternal antibody could not provide pertinent protection against the prevalent strains [ ] . furthermore, there was accumulating evidence indicated that the nephropathogenic ibvs have become prevalent in china in last several years [ , , ] . through clinical records and the virus recovery trials, identified isolates mainly caused typical swollen kidney, different from the respiratory type strains isolated in earlier years, including the major vaccine strains. these findings indicated that all isolated ibv strains from china during - were evolutionarily distant from the vaccine strains used for current, resulting in vaccination failure cases. the s protein determined the serotypic evolution, the phenotype change and the genetic diversity of ibvs [ ] . in the present study, nucleotide and derived amino acid sequences of s protein genes of the field strains were aligned and compared to the representative strains, to determine the relationship of circulating field isolates, vaccine strains and previously described variant strains. newly isolated strains shared between . % to % nucleotide sequence similarity with each other, higher similarity than the vaccine strains and other representative ibvs. although the ibvs all over the world shared some common antigenic types, virus strains within a geographic region were unique and distinct, even in different provinces of china. the variants were mostly located in the first amino acids in the n-terminal of the s protein of ibv, even though the mutants consisted of insertions, deletions and point mutations were complicated and detailedly different, the hypervariable regions in s protein in this study were similar to previous studies [ , , ] . the phylogenetic analysis showed that there were five subgroups of ibvs co-circulating in china, and multiple strains might cause the constant ib outbreaks. the newly isolated strains were mostly derived from a , / and hn . only ck/ch/chongqing/ belonged to the branch of gray. the phylogenetic distributions were closely relative to geographical factors. most of the recently isolated ibvs in this study formed the distinct cluster related to the a type. however, the routine vaccine strains mainly belong to m -type branch. a strain is closely related to / serotype, spreading over europe since its first isolation in uk in [ , , [ ] [ ] [ ] [ ] [ ] . in this study, . % ( / ) field isolates belonged to the a type branch, which included . % ( / ) nephropathogenic field isolates of this study. the qxibv, first isolated in china and reported associated predominantly with various forms of renal pathology in china, was also representative a -type strain [ , ] . the analysis results were according to the prevalence of nephropathogenicity ib. to date, the qx-like ibv strains have been widely isolated in many european countries, and become a dominant genotype [ , ] . through ib surveys, the european qx-like ibv strains have been reported that caused % respiratory signs, % litter or enteric problems, only % had swollen kidneys [ ] . absorbingly, the qx-like ibv strains have undergone divergent evolution paths, brought out different variants in europe and china. similarly, seven exceptional strains located in the a -type branch caused evident respiratory problems, including three isolates from zhejiang province (qz , qz and qz ) and three isolates from guangdong province (xd , xd and lz ), and gl from guangxi province. the results of our study indicated the strain grouping, such as phenotype and genotype, were not only depended on the geographical factors. the evolutionary pace and the epidemiology characteristics of the ibv were complicated. in conclusion, the data obtained from our study suggest most of present ibv isolates in china are a -like nephropathogenic strains. to control the prevalence and well prepare for the potential outbreaks of ib, the candidate virus strain for vaccination might be selected timely and specifically in a geographical region, which manifests the importance of continuing surveillance of new ibv strains. this paper is a periodic report on our ongoing surveillance program. we hope the study could contribute to guiding the development of effective vaccines and establishment of control policy for ib. during the period from june to november , circulating field ibv isolates were selected from suspected broilers and broiler breeders in vaccinated flocks from eastern, southern, southwestern and central china. documented clinical signs of the birds included typical the allantoic fluids containing ibv isolates after h post inoculation were harvested for subsequent experiments, and the remains were preserved in liquid nitrogen. five -day-old spf white leghorn chickens were intranasally inoculated with filtration sterilized allantoic fluid of each isolated virus strains, respectively. all of the chicks were examined and recorded daily for clinical signs of infection and mortality for days post-inoculation, the dead birds were necrospied for lesions of respiratory tract or nephritis. finally, all the survivors were sacrificed and necrospied. rt-pcr and s gene sequencing a pair of specific primers was designed to amplify the entire s protein gene, including the forward primer (s f): '-aagactgaacaaaagaccgact- ', and the reverse primer (s r): '-caaaacctgccataac-taacata- '. reverse transcription and amplification were performed using the primescripttm one-step rt-pcr kit in μl reaction volume containing μl of rt-pcr premix (reaction buffer, dntps, μl of enzyme mix), μl of extracted viral rna and the specific primer pair. reverse transcription and amplification were performed as one cycle of °c for min, °c for min, followed by cycles of denaturation at °c figure the phylogenetic tree of ibvs isolated in mainland china during - based on the viral s sequences. the reference strains in this study were marked with "▲". for s, annealing at °c for s and extension at °c for min, respectively) with a final min extension step at °c. the pcr products were cloned into pmd -t vector (takara biotechnology, dalian, china) for later sequencing (augct biotechnology, beijing, china). the s protein gene sequences obtained in this study were submitted to the genbank database and assigned the accession numbers of gu -gu , gu -gu (table ) . twenty-eight representative sequences available in genbank were contributed to comparison and phylogenetic analysis in this study, including vaccine strains, h (accession numbers: . the multiple-alignment was carried out using dnastar sequence analysis software (dnastar inc., madison, wi, usa). the phylogenetic tree was constructed using the mega . software with neighbor-joining method and each tree was produced using a consensus of bootstrap replicates [ ] . a nomenclature for avian coronavirus isolates and the question of species status infectious bronchitis protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus the experimental infection of chickens with mixtures of infectious bronchitis virus and escherichia coli significance of interactions between escherichia coli and respiratory pathogens in layer hen flocks suffering from colibacillosisassociated mortality cloning and sequencing of genes encoding structural proteins of avian 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nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china genotypic and phenotypic characterization of the california (cal ) variant of infectious bronchitis virus co-circulation of four types of infectious bronchitis virus ( /b, /i, b and massachusetts) variation in the spike protein of the /b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs a survey of the presence of a new infectious bronchitis virus designated / ( b) a new strain of infectious bronchitis virus infecting domestic fowl in great britain epidemiology of infectious bronchitis virus in belgian broilers: a retrospective study pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma and / serotypes a reverse transcriptase polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from mega : molecular evolutionary genetics analysis (mega) software version . phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during authors' contributions jj and jx carried out most of the experiments and wrote the manuscript, and should be considered as first authors. fc and qx critically revised the manuscript and the experiment design. ds, kz, cx, jq, hl, jm and yb helped with the experiment. all of the authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- -i cywew authors: pfefferle, susanne; krähling, verena; ditt, vanessa; grywna, klaus; mühlberger, elke; drosten, christian title: reverse genetic characterization of the natural genomic deletion in sars-coronavirus strain frankfurt- open reading frame b reveals an attenuating function of the b protein in-vitro and in-vivo date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: i cywew during the outbreak of sars in / , a prototype virus was isolated from a patient in frankfurt/germany (strain frankfurt- ). as opposed to all other sars-coronavirus strains, frankfurt- has a -nucleotide deletion in the transmembrane domain of its orf b protein. when over-expressed in hek cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase . to study the role of orf b in the context of virus replication, we cloned a full genome cdna copy of frankfurt- in a bacterial artificial chromosome downstream of a t rna polymerase promoter. transfection of capped rna transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. the presumed frankfurt- ancestor with an intact orf b was reconstructed. in caco- and huh cells, but not in vero cells, the variant carrying the orf b deletion had a replicative advantage against the parental virus ( - and -fold increase of virus rna in supernatant, respectively). this effect was neither associated with changes in the induction or secretion of type i interferon, nor with altered induction of apoptosis in cell culture. however, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain ( . -fold in vero cells, . -fold in caco- cells). in syrian golden hamsters inoculated intranasally with e plaque forming units of either virus, mean titers of infectious virus and viral rna in the lungs after h were increased - and . -fold, respectively, with the deleted virus. this difference could explain earlier observations of enhanced virulence of frankfurt- in hamsters as compared to other sars-coronavirus reference strains and identifies the sars-cov b protein as an attenuating factor with the sars-coronavirus genome. because attenuation was focused on the early phase of infection in-vivo, orf b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the sars epidemic. in syrian golden hamsters inoculated intranasally with e plaque forming units of either virus, mean titers of infectious virus and viral rna in the lungs after h were increased -and . fold, respectively, with the deleted virus. this difference could explain earlier observations of enhanced virulence of frankfurt- in hamsters as compared to other sars-coronavirus reference strains and identifies the sars-cov b protein as an attenuating factor with the sars-coronavirus genome. because attenuation was focused on the early phase of infection in-vivo, orf b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the sars epidemic. the severe acute respiratory syndrome (sars) emerged in the end of in china and caused an international epidemic [ ] . its causative agent, a hitherto unknown coronavirus (cov) is thought to have been circulating in an animal reservoir before it crossed species barriers into humans [ ] [ ] [ ] [ ] [ ] [ ] . bats have been implicated as the original reservoir of all cov, and the large range of relevant human and animal cov has been suggested to be resulting from host switching events [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the context of viral host switching, it is interesting that several sars-cov proteins encoded on subgenomic (sg) rnas seem to be dispensable for virus replication in cultured cells of primate or rodent origin, as well as in rodent models [ ] [ ] [ ] . because these orfs are not shared between different cov groups, they are referred to as group-specific orfs [ ] . proteins encoded by group-specific orfs have been shown to influence pathogenesis, virus replication, or host immune response [ , [ ] [ ] [ ] [ ] [ ] . during the human sars epidemic, sars-cov has rapidly acquired deletions in several of its group-specific orfs [ , [ ] [ ] [ ] . the original functions of associated proteins might exemplify mechanisms through which highly pathogenic zoonotic viruses such as the sars-cov can persist in their reservoirs without causing disease. the characterization of virus proteins can be unreliable if only the protein of interest is studied on its own. the study of proteins in the whole virus context reflects virushost interactions more realistically, and takes into account intraviral protein interactions. such experiments can be done using reverse genetics techniques which for most plus-strand viruses rely on cloned cdna copies of the whole rna genome that can be mutagenized in-vitro [ ] [ ] [ ] . different approaches have been followed to implement cov reverse genetics. a great challenge in this regard is the huge size of the cov genome, making cloning procedures difficult because plasmid-based cdna constructs are instable in e. coli. in-vitro ligation of subgenomic cdna fragments without the assembly of full-length plasmids has been successfully used to establish cov reverse genetics [ ] [ ] [ ] . as an alternative, full-length cdna copies have been reconstructed and kept in vaccinia virus [ , ] . a third approach is based on bacterial artificial chromosomes (bac) for keeping full-length cov cdna stable, owing to a low copy number of bac dna per e. coli cell [ ] [ ] [ ] [ ] . the first two systems use t rna polymerase promoter-driven in-vitro transcription of capped, infectious rna that is transfected into cells. the latter uses a cmv promoter and relies on the transfection of full-length cdna into cells, which is then transcribed in the nucleus into infectious rna. in this study we have implemented a modified approach to cov reverse genetics by cloning the entire sars-cov genome downstream of a t rna polymerase promotor in a bac. using the linearized bac construct as a template for in vitro transcription, this system combines plasmid-based handling of cdna constructs with direct delivery of genome-like rna into the cytoplasm. the novel system was used to characterize a nucleotide in-frame deletion in orf b that is present in the primary isolate of sars-cov prototype strain frankfurt- [ ] . this specific deletion is not present in any other of > sars-cov orf b sequences in genbank, and in none of the sars-like bat cov. however, deletions of the whole orf b and beyond have been acquired by sars-cov during the sars epidemic in humans [ ] [ ] [ ] . the orf b protein is a amino acid protein that is transcribed by a noncanonical leaky scanning mechanism from the second orf encoded on subgenomic rna [ , ] . the protein is a type iii integral transmembrane protein located in the golgi compartment [ ] . it has been shown previously that the protein is a structural virion component, that it is dispensable for replication in various cell cultures, and that it induces apoptosis in cultured cells if overexpressed [ , ] . the pro-apoptotic effect seems to be limited to late stages of the apoptotic cascade [ ] . qualitatively the same effect was confirmed in studies on a recombinant virus, containing a combined deletion of orf a and orf b [ ] . however, it is unclear to what extent either the orf a or orf b proteins, respectively, contribute to the effect. it is also unclear to what degree the orf b protein alone influences virus replication in-vivo. this is relevant for the frankfurt- virus because it has been used as a model virus in several studies on pathogenesis and antiviral drug research (e.g [ ] [ ] [ ] [ ] ). finally it is unclear whether the frankfurt- orf b deletion has been acquired during cell culture, or whether it may have been present already in the patient and may have undergone transmission. in this study, primary clinical samples from the frankfurt index patient and a secondary case who acquired her infection from him were re-analyzed. frankfurt- viruses with and without the deletion were then reconstructed by reverse genetics. effects of the deletion on interferon induction and response, on induction of apoptosis, and on in-vivo replication in syrian golden hamsters were determined. the frankfurt- sars-cov cell culture isolate contained a nt in-frame deletion within a predicted transmembrane region. a back-translated blast search on the nucleotide database (tblastn) showed that this deletion was not present in any of > sars-cov orf b sequences in genbank (except in an independent sequence of the frankfurt strain), and in none of sarslike bat-cov sequenced in the orf b region (figure ). to determine whether the deletion originated from the infected patient or was generated in cell culture, rt-pcr was used to screen for the deletion in several sequential samples from the frankfurt index patient of whom the frankfurt- isolate had been taken. as shown in figure , all patient samples yielded dna bands of higher molecular weight than those from the cell culture isolate, indicating absence of the deletion in the patient. of note, clinical samples from the wife of the index patient, who got infected by her husband in later course, did not contain the deletion either ( figure ). to exclude that a minor background of the virus population in patient samples might have carried the deletion already prior to virus isolation, a second pcr was done with a primer bridging the deleted region (i.e., it bound up-and downstream of the deletion and would only amplify deletion-containing viruses). the deleted virus could not be detected in any patient sample. it was therefore assumed that the virus had acquired the deletion during isolation in cell culture. several sars-cov accessory gene products have been shown to be involved in the induction of apoptosis, including the a and b proteins [ , , ] . to analyze whether the deletion in orf b had any influence on its amino acid variability in orf b and rt-pcr analysis of orf b in clinical samples versus cell culture isolate figure amino acid variability in orf b and rt-pcr analysis of orf b in clinical samples versus cell culture isolate. (a) orf b amino acid alignment of all sars-and sars-like cov available in genbank (sequences yielding identical alignments in the region of interest were deleted). the transmembrane domain [ ] is shaded in black/grey. the left column shows gen-bank accession numbers of representative genomes for each unique amino acid sequence, along with the starting nucleotide positions of orf b in each genbank entry. the right hand column shows strain designations and their sources (human, civet, bat). only one sequence derived from the frankfurt- strain (ab ) shows a nucleotide in-frame deletion in the predicted transmembrane domain (tmd [ ] . lane shows the corresponding amplification product in the original sputum sample that yielded the frankfurt- isolate. lane depicts the pcr product of the virus isolate derived from this sample. ability to induce apoptosis, vero e cells were transfected with expression plasmids encoding orf a, orf b or orf b del containing a deletion exactly corresponding to that in frankfurt- . control cells were infected with sendai virus (sev) or left untreated. forty-eight hours later, lysates were analyzed for procaspase cleavage by western blot using an antibody that detects cleaved and non-cleaved forms. as shown in figure a , cleavage of caspase was observed in cells expressing orf a and orf b. interestingly, expression of orf b del did not cause caspase cleavage. to examine the effect of the orf b deletion on the type i ifn response, reporter gene assays were performed. cells were transfected with plasmids encoding orf a, orf b or orf b del, respectively. all cells were co-transfected with the phisg- reporter plasmid containing the firefly luciferase gene under the control of the isre region of the human ifn-stimulated gene . expression plasmid prl-sv encoding renilla luciferase was co-transfected to normalize for interferon-independent stimulation of transcription. twenty-four hours later, the cells were infected with sev to induce ifn-mediated reporter gene expression. cells were lyzed h post infection and subjected to reporter gene assays. as shown in figure b , expression of both orf a and orf b but not orf b del induced ifn-dependent reporter gene expression. in those cultures superinfected with sev, none of the plasmids reduced the sev-associated, ifn-dependent reporter gene expression. the ebolavirus vp , a known antagonist of interferon induction, clearly showed reduction of reporter gene expression if used in the same system ( figure b ) [ , ] . these distinct findings prompted us to elucidate b protein functions in the natural virus context. to be able to measure even marginal phenotypical differences we decided to reconstruct both genotypes while establishing a novel reverse genetic system. in order to clone subgenomic portions of the sars-cov genome, seven pcr fragments covering the whole genome were generated with primers described by yount et al. [ ] . fragments were initially cloned in high copy number plasmid vectors, or, if refractory to cloning, in low copy plasmids as shown in figure . except some marker mutations (see below), the sequence of cdna inserts in the seven resulting subclones was corrected to match that of the cell culture-derived virus by plasmidbased inverse pcr and fragment-extension pcr. for construction of the variant with an intact orf b, the nt deletion was filled in by oligonucleotide extension pcr on subclone pf ( figure ). corrected subclones were assembled in a stepwise procedure into four bac clones containing about a quarter of the sars-cov genome each, which where then joined into a full length bac cdna clone (refer to figure and the materials and methods section for more details on the construction). bacs containing both versions of subclone f were assembled. both bacs were sequenced, confirming presence of all marker mutations and absence of any further mutations (refer to influence on apoptosis and type i interferon induction by overexpression of orf a, orf b, and orf b with the frankfurt- -specific deletion interferon beta promoter-specific reporter gene expression (y-axis), shown as the factor of induction as compared to the mock-transfected, non-superinfected control (see below). the assay was done by transfection of hek cells with plasmids expressing either ebolavirus vp , orf a, orf b, or orf b with a deletion corresponding to the orf b deletion in frankfurt- (x-axis), as well as reporter constructs for the interferon beta promoter (firefly luciferase) and the sv promoter (renilla luciferase). h post transfection, cells were either superinfected with sev ( hemagglutinating units) or left uninfected. interferon-specific reporter gene expression was determined h after superinfection (black bars) or mock infection (grey bars). the experiment was done in triplicate and standard deviations are shown. to determine interferon-specific expression, the firefly luminescence signal was divided by the renilla luciferase signal. genbank accession number fj ). one whole bac was digested with bgl i, which was present at seven positions on the bac construct. as shown in figure a , fragments of the expected sizes were obtained. the linearized bac cdna and a pcr product containing the nucleocapsid gene were in-vitro transcribed and cotransfected in bhk cells. because bhk cells did not support sars-cov replication, supernatants were transferred assembly of a full-length sars-cov cdna clone in a bac (refer to materials and methods section for a detailed description of construction steps) figure assembly of a full-length sars-cov cdna clone in a bac (refer to materials and methods section for a detailed description of construction steps). (a) arrows symbolize positions of pcr fragments on the sars-cov genome. these were cloned in subgenomic plasmids. (b) subgenomic plasmids pa pf. plasmids are either based on psmart (identified by an "s" symbol within the respective clones) or on pcr . (no symbol). squares on each plasmid symbolize the approximate positions of erroneous mutations from initial cloning corrected by fragment-extension technique before assembly to higher-order clones. small extension-pcr symbols above clones pb and pf indicate mutations introduced into plasmids to facilitate subsequent construction steps (deletion of an mlu i-site in pb) or to fill in the nt deletion in orf b in pf. (c) assembly of quarter clones. circles represent plasmids, ovals represent bacs. bold grey arrows symbolize essential bacencoded genes reconstituted during bac ligation, in order to achieve high cloning efficiency. restriction digestion steps are symbolized by thin arrows. the utilized restriction enzymes are identified next to the arrows. pcr primer symbols (small arrows) next to plasmids indicate that these plasmids were first amplified with primers introducing restriction sites (identified next to primer symbols) before the resulting products were double-digested as indicated. the large horizontal arrows below plasmids pa and pa indicate that these fragments were joined by overlap-extension pcr with primers eliminating a bgl i restriction site as symbolized by a square on both of the parental plasmids. in each construction, fragment ends shown in close proximity were first ligated in-vitro. the ligation products were then purified, ligated at sites drawn in greater distance, and transformed in e. coli. to vero cells susceptible for sars-cov infection. virus progeny was identified by immunofluorescence analysis with anti-sars-cov patient serum after h ( figure b ), as well as by plaque assays after h ( figure b ). electron microscopy showed intracellular structures compatible with sites of virion assembly as well as mature virus particles ( figure c ). the recombinant virus containing the full-length orf b gene was named rscv. the virus containing the deletion in orf b was termed r bΔtmd. both viruses were amplified on vero cells and stored for further experiments. to confirm the purity of virus preparations, two different rt-pcr assays were done. the first assay utilized primers spanning the deletion in orf b, as shown in figure a. both preparations yielded singular pcr products whose molecular weight was lower for r bΔtmd than for rscv. the molecular weight difference corresponded to the size of the orf b deletion. for confirmation, a second rt-pcr assay was done with a primer hybridizing with the deleted portion of orf b that was missing in r bΔtmd. a singular band was obtained for rscv but not for r bΔtmd ( figure a ). identity of all pcr products was confirmed by sequencing. since an appropriate antibody directed against orf b was not available when we started these studies, a ddddk (flag-) tag sequence was introduced in the infectious clone prscv by overlap-extension pcr at the c-terminus of orf b. as shown in figure a , a protein band corresponding to the predicted molecular weight of the b protein ( . kda) was specifically detected in rscv bflaginfected cells using an anti-flag antibody. also, immunofluorescence analyses revealed a dotted perinuclear pattern in rscv bflag-infected cells stained with an anti-flag antibody, whereas rscv-infected cells incubated with the same antibody did not show fluorescence ( figure b ). expression of the nucleocapsid (n) protein was confirmed with a human serum directed mainly against n with both viruses ( figure b ). it was concluded that the orf b protein of the recombinant viruses was expressed in infected cells, and that its principal properties are not affected by a c-terminal flagtag epitope. these findings, including the pattern of fluorescence when expressing orf b, were consistent with earlier reports by pekosz et al [ , ] . growth properties of rscv and r bΔtmd on different cell lines were compared. plaque morphology was determined for both viruses, with no discernible differences ( figure b ). because plaque assay could only show cells that die from virus infection, the same experiment was repeated and read out by immunofocus assay, using serum of a human sars survivor. there was no difference in immunofocus morphology ( figure b ). growth curves in three different cell cultures were determined next. virus rna was measured in supernatant by real-time rt-pcr. a multiplicity of infection (moi) of . was used for both recombinant viruses in vero and caco- cells. for huh cell, an moi of . was used, due to their lower susceptibility to sars-cov infection. in vero cells, very similar increases in rna concentration were observed with both viruses during hours ( figure c ). in caco- and huh cells, respectively, r bΔtmd accumulated about -and -fold more rna than rscv. it was concluded that the deleted virus had a slight but reproducible growth advantage in the latter cell lines. in the absence of mechanisms of adaptive immunity, replication of rna viruses is controlled by production of and response to type-i interferons, as well as apoptosis of infected cells. taking into account our findings in overexpression experiments, central elements of these systems were therefore examined in cells infected with both virus variants. because vero cells as well as huh- cells are deficient in interferon induction [ ] , hek -lp cells were used to analyze interferon beta mrna transcription. these cells have been shown to be capable of inducing and secreting interferon, and they are susceptible to sars-cov infection [ ] . hek -lp cells were seeded in six-well plates and infected with rscv or r bΔtmd at an moi of . as shown in figure a , infection with the control virus ndv elevated the transcription level of interferon beta mrna by a factor of . rscv did not induce interferon beta mrna transcription, confirming earlier findings [ ] . induction of transcription was not observed with r bΔtmd either, indicating that the orf b protein is not involved in the comparison of recombinant viruses rscv and r bΔtmd rscv is the full-length orf b virus; r bΔtmd is the virus with the frankfurt- -specific deletion in orf b as shown in figure . (b) plaque assay using crystal violet stain and immunofocus assay using a polyclonal protein patient serum reacting predominantly against the n protein (anti-n). (c) relative log rna concentration (copies per ml) in viral supernatants after growth in cell lines as indicated. the zero value on the y-axis represents the starting rna concentrations after virus absorption ( h) and change of medium in each culture. other data for each culture were normalized by subtraction of the logarithmic starting concentration. each datum point shows the mean value of three independent experiments. expression of orf b ablation of interferon induction conferred during sars-cov replication. essentially the same results were obtained with caco- cells ( figure b ). hek -lp cells were used to study release of interferon alpha in the supernatants of infected cells. it has been reported by spiegel et al. that interferon alpha expression is induced in sars-cov-infected -lp cells to a certain level [ ] . exactly the same cells were obtained from f. weber, university of freiburg, and interferon alpha transcription after infection with sars-cov was qualitatively confirmed by rt-pcr in our laboratory (not shown). the level of interferon alpha was then determined by eia in supernatant of -lp cells, h after infection of both viruses at an moi of . as shown in figure a , infection with the control virus ndv elevated the interferon alpha level in supernatant by a factor of , while neither rscv nor r bΔtmd caused detectable secretion. to study the effects of interferon on replication of both viruses, vero cells were pre-treated with increasing concentrations of interferon beta in order to induce an antiviral state. cells were infected with either rscv or r bΔtmd at an moi of . . as shown in figure b , r bΔtmd replicated to marginally higher virus concentrations than rscv in presence of interferon (up to . fold increase). since in our hands caco- cells were more resistant to interferon beta pre-treatment than vero cells, experiments were repeated with higher concentrations of interferon using caco- cells. more efficient replication (up to . fold increase) was again observed for r bΔtmd ( figure b ). programmed, caspase-mediated death of infected cells is an efficient way of controlling virus replication. several sars-cov accessory gene products have been implicated in the induction of apoptosis, including the orf a and orf b proteins as confirmed in this study (figure ). activation of apoptosis was therefore compared in cells infected with either rscv or r bΔtmd. vero cells were infected at an moi of of either virus and assayed by western blot for activation of caspase , the central element of the apoptosis induction cascade. as opposed to the clear effect seen in overexpression experiments ( figure ), both viruses induced partial cleavage of procaspase at hours post infection, and complete cleavage after hours (figure ). to confirm these results we analyzed cleavage of poly-adp ribose polymerase type (parp- ), a downstream effect of caspase- activation [ ] . as shown in figure , western blot showed little differences in processing of parp- in vero cells with both viruses. it was concluded that the deletion-dependent ablation of the pro-apaptotic effect of orf b as observed in overexpression experiments was irrelevant in the context of full virus replication in cell culture. deletions in and around the sgrna region occurred during the epidemic and were transmitted in the community [ ] [ ] [ ] . in order to elucidate whether the orf b deletion might influence replication in-vivo, both viruses were tested in hamsters. syrian golden hamsters have been shown to be an acceptable rodent model for sars-cov replication and pathogenicity [ , ] . four groups of three hamsters were infected via the intranasal route with pfu of either rscv or r bΔtmd, and sacrificed on day or , respectively. whole lungs were minced and tested for infectious virus and viral rna. the deleted virus yielded -fold more infectious particles and -fold more rna copies in the lungs on day (figure and table ). differences decreased but remained qualitatively equivalent by day ( -fold and . -fold more infectious virus and rna, respectively). the differences in rna concentrations were borderline significant on day (table ) . t-tests did not identify further significant differences between our small groups of animals, and we did not want to use more animals for these experiments. in one of three animals sacrificed on day post infection, rscv failed to replicate entirely ( figure ). the replication advantage for r bΔtmd was in concordance with findings in caco- and huh- cell cultures ( figure ). in the present study we have characterized a naturallyacquired deletion in the orf b of the primary sars-cov frankfurt- isolate by reverse genetics. in contrast to other plus strand rna viruses it has taken rather long to complete the first coronavirus reverse genetics systems [ , , , , , ] . it has been difficult to clone complete cov genomes due to their large sizes and toxicity or lability of constructs in e. coli [ , , ] . this has been circumvented by baric et al. by the use of subgenomic plasmids that are ligated in-vitro to full genomic cdna, prior to transcription and electroporation [ ] . we tried this approach initially, but we failed to generate sufficient amounts of full-length cdna for in-vitro transcription. thiel et al. have described an approach to generating fulllength cdna by stepwise assembly of an entire coronavirus genome in a pox virus backbone [ ] . as we had not worked with pox viruses before, this technique appeared rather difficult to establish. as a third alternative, enjuanes and coworkers have presented an approach based on cloning of the entire genome in bac and transfecting the bac-contained viral cdna under the control of a cmv promoter [ ] . the use of bac dna provides the remarkable benefit of being able to handle full length genomic dna in one plasmid backbone, using standard dna cloning techniques. as demonstrated in several studies of that group [ , , , [ ] [ ] [ ] , bac manipulations are rather fast and straightforward, while providing little opportunity for de-novo mutations resulting from dna manipulation steps. in our strategy we used a bacteriophage t -derived rna polymerase promoter instead of the cmv promoter because we wanted to provide a genome that most closely resembled that of the virus, using cytoplasmic sites for replication and circumventing transcription and possible splicing in the nucleus [ , , ] . a t promoter has not been used before with a plasmid-contained cov cdna genome; it was conceivable that leaky transcription might enhance underlying toxicity of cov genomes in e. coli. our study shows that the sars-cov genome is stable in bac despite the t promoter. interestingly, enjuanes and colleagues have made bac-based full length clones for different cov and reported that their sars-cov bac clone was more stable than, e.g., the one they developed for tgev [ ] . the sars-cov genome may thus be more stable in e. coli than that of other covs. it remains to be seen whether combined t /bac infectious cdna clones can also be constructed for other covs. the nucleotide in-frame deletion in the transmembrane domain of orf b is a paramount feature of the frankfurt- strain. this strain has been employed as a prototypic sars-cov in several studies on pathogenesis and antiviral therapy (e.g., [ ] [ ] [ ] [ ] ). by analysis of primary clinical samples from the patients treated in for sars in frankfurt, we could show that the mutation has been selected during initial isolation in cell culture, and that it did not stem from the frankfurt index patient [ ] . initial characterizations of the protein by overexpression experiments suggested reduced induction of interferon and apoptosis in association with the deletion, which led us to reconstruct the corresponding viruses with and without the deletion by reverse genetics. in concordance with earlier findings, type i interferon was neither induced nor produced by either sars-cov variant in our study [ , [ ] [ ] [ ] [ ] . it is assumed that cov either encode a range of proteins interacting with interferon sensing, or shield their rna from immune recognition through the formation of double membrane vesicle-based replication compartments [ , [ ] [ ] [ ] . our experiments suggest that orf b is not necessary for sars-cov counteraction against the induction of the interferon beta promoter. it also seems unlikely that orf b contributes to the interference of sars-cov with secretion of interferon alpha [ ] . however, the deleted virus showed slightly decreased sensitivity to pretreatment of cells with interferon. this effect was remarkable since earlier studies only determined opposite (= evasive) effects on the interferon response for cov accessory proteins. these include interference with the interferon signalling cascade in the case of sars-cov protein , or prevention of activation of interferon-sensitive induction of apoptosis by recombinant coronaviruses rscv and r bΔtmd in-vivo effect of the orf b deletion figure in-vivo effect of the orf b deletion. golden syrian hamsters were infected with pfu of rscv and r bΔtmd (x-axis). heat inactivated rscv served as mock control. for each point of time post infection, three animals per virus variant were sacrificed (animals , , as identified on the xaxis). lungs were taken in total. viral titers were determined by plaque assay and viral rna was quantified by real-time rt-pcr. light grey bars represent log copies of viral rna, dark grey bars represent pfu per g lung tissue. the arrow indicates one animal with failure of virus replication. genes for mouse hepatitis virus nucleocapsid protein [ , ] . here we observed an orf b-dependent extension of the replication-attenuating effect of interferon. however, the additional extent of attenuation on top of the effect of interferon beta was of the same size as that observed in untreated cell cultures (compare figure and figure ) and did hardly increase with increasing interferon concentrations. this suggests an additive rather than a synergistic effect of orf b and interferon on the attenuation of virus replication. in spite of the high relevance of the interferon response for controlling sars-cov replication, we should therefore assume that orf b plays no role in the context of the type i interferon system [ , ] . and in-vivo. our initial overexpression experiments pointed to a strong pro-apoptotic effect of intact orf b, which was in concordance with a study by schaecher et al. who found that sgrna -derived proteins activated caspase if overexpressed [ ] . complementary to their study, however, our experiments did not confirm any similar effect specifically for the orf b protein in the full virus context. schaecher et al. studied a recombinant virus with a double deletion of both orf a and b, and this virus induced apoptosis clearly less efficiently than the parent full-length virus [ , ] . the most likely explanation for the difference between both viruses is that the pro-apoptotic effect of gene proteins observed by schaecher et al. was contributed by orf a rather than orf b. even though the orf b deletion in frankfurt- was not affecting interferon and apoptosis systems, the virus with a deletion seems to have been selected during isolation in cell culture and shows a replicative advantage in two of three cell lines. this is remarkable because sars-cov variants with deletions in the orf (and also orf ) gene region have been transmitted and maintained in humans in the late phases of the epidemic [ ] [ ] [ ] . it has never been formally addressed whether these viruses might have undergone particularly efficient transmission. we therefore determined whether the orf b deletion in frankfurt- conferred a replicative advantage in-vivo, using syrian golden hamsters as a model of human sars-cov infection [ , ] . interestingly, the enhancing effect of the orf b deletion was even more pronounced in hamsters than in cell culture. hamsters infected with the deleted variant had significantly more virus rna and a fold increase of infectious virus titers in their lungs after h. the rate of successful infections was / with the deleted virus and / with the full virus. in concordance with these observations, roberts et al. have described ca. -fold more efficient replication of frankfurt- in hamsters as compared to urbani and hku- [ , ] . mortality in hamsters was only observed with frankfurt- ( of animals) but not urbani and hku- [ , ] . it was suggested that an amino acid exchange (l f) in the s -domain of the spike protein of frankfurt- against both urbani and hku- might explain the difference. however, a replicative difference in extent similar to that reported by roberts et al. was observed in our study between two variants of frankfurt- that differed only by the orf b deletion. as the deletion is not present in urbani or any other prototype strain, this identifies the b protein as a potential attenuating factor within the genome of sars-cov. we have seen in this study that the attenuating effect of orf b was focused on the early phase of infection invivo. because it has been suggested that delayed accumulation of high virus concentrations in infected patients has limited the spread of sars-cov in the population, it is tempting to speculate that the occurrence of viruses with deletions in the orf / region in the late phase of the epidemic might have added to the efficiency of virus transmission in humans [ ] [ ] [ ] . it will be interesting in the future to investigate the exact mechanism of orf bdependent attenuation, and to determine whether this might contribute to the maintenance of virus in its natural reservoir. the original vero cells on which frankfurt- was primarily isolated (hereafter termed vero fm, obtained from jin- plaque assays were done with avicel overlays (rc , fmc biopolymer, belgium) as described elsewhere [ ] . immunofocus assay used the same overlay and was otherwise performed as described previously [ ] . viral rna quantification using in-vitro transcribed rna standards was done as described previously [ ] . standard cloning techniques were used. all gel purifications were done with the qiaex ii kit (qiagen, hilden, germany). dna constructs were electroporated into e. cloni (lucigen, middleton, usa) or stbl e. coli cells (invitrogen, karlsruhe, germany). prior to digestion with methylation-sensitive endonucleases plasmids were transformed in sure cells (stratagene, la jolla, usa). bac preparations were done with the nucleobond ® ax-kit (macherey nagel, germany) as instructed. plasmid-based inverse pcr was performed with quikchange xxl kit (stratagene, usa). pcr mutagenesis by overlap-extension pcr used phusion ® dna polymerase and around ng of input plasmid dna. sars-cov coding sequence within constructs was fully sequenced after every mutagenic step. total rna was extracted from infected vero cells with the qiagen rneasy kit. using primers described by yount et al. [ ] , cdna fragments spanning the sars-cov genome were generated by rt-pcr using superscript iii reverse transcriptase and expand high fidelity dna polymerase mixture. these primers inserted bgl i restriction sites at fragment borders and a t promoter in front of the 'end of the genome [ ] . in addition to the strategy described by yount et al., a not i restriction site was introduced downstream of the genomic poly-a tail. figure gives an overview of cloned fragments. fragment a was cloned in two parts (a and a , figure the fragments were overlap-extended, digested with bamh i and not i, and cloned back into the corresponding restriction sites in clone pf. all clones were verified by sequencing. using the same technique, a ddddk (flag-) tag sequence was introduced at the c-terminus of orf b, with overlap-extension primers '-gattacaaggatgac-gacgataagtaaacgaacatgaaacttctc- ' and '-cttatcgtcgtcatccttgtaatcgactttggtacaag-gttct- '. assembly of full length bac cdna clone bac vector pbelobac was obtained from neb, boston, usa. the nco i site at position was oblated by primer extension mutagenesis, resulting in pbelodnco. the not i-not i multiple cloning site fragment was removed from pbelodnco and replaced by an oligonucleotide adapter containing nsi i, bsah i, sph i and not i restriction sites in sequence, resulting in pbeload . fragment a was amplified from plasmid pa with primers '-agtaatgggccctaagtactaatacgactcactataga-tattagg- ' and '-acaccatagtcaacgatgcc- ', thereby introducing a pspom i site upstream of the t promotor (figure ). the fragment was digested with pspom i and bgl i and ligated to the long ecor i not i fragment of pbeload (pbeload a, figure ). the '-most , nt were amplified from plasmid pb using primers '-gcctatatgcatggatgttagag- ' and '-atgaat-gcggccgctacactcaacacgtgtggcacgc- ', thereby introducing a not i site immediately downstream of the mlu i site at position . the pcr product was digested with bgl i and not i, gel purified, and ligated to the dephosphorylated short not i ecor i fragment of pbeload (pbeload b , figure ). pbeload a and pbeload b were gel purified and ligated, resulting in quarter clone pab, (figure ). the '-most , nt were amplified from plasmid b in two parts, using primers '-tagactacgccggcg-tagccttaggtttaaaaacaattgccactc- ' and '-tacactcaacacgtgtggcacgattgcgct- ' ( '-part); and primers '-agcgcaatcgtgccacacgtgttgagt-gta- 'and '-tgaaccgccacgctggctaaacc- ' ( 'part), respectively. both products were overlap-extended, resulting in a pcr product with a depleted mlu i site at position . the product also contained a not i site upstream of the bsu i site at position , introduced by a primer '-overhang. the product was not i and bgl i digested and ligated to the long ecor i not i fragment of pbeload (pbeload b , figure ). plasmid pd was digested with bgl i and bcl i (compatible to bamh i). the fragment was ligated to the dephosphorylated short bamh i ecor i fragment of pbelodnco (pbeloncod , figure ). fragment c was cut out of its psmart vector with bgl i and dephosphorylated, followed by ligation to pbeloncod and gel purification. this product was ligated to pbeload b , generating quarter clone pbcd. the acl i bgl i fragment of vector d- - was ligated to the long ecor i bsah i fragment of pbelodnco (bsa hi is compatible with acl i) (pbeloncod , figure ). the dephosphorylated pst i bgl i fragment of vector pe was ligated to the short nsi i ecor i fragment of pbeload (nsi i is compatible with pst i) (pbeload e , figure ). this product was ligated with pbeloncod to yield quarter clone pde. the , bp sph i bgl i fragment of subclone pe was ligated to the long ecor i sph i fragment of pbeload (pbeload e , figure ). the bgl i not i fragment of plasmid pf was ligated to the short not i ecor i fragment of pbeload (pbeload f, figure ). this fragment was ligated with pbeload e , yielding quarter clone pef. quarter clones pab and pbcd were digested with bsu i and pspom i, the latter cut destroying the replicative element sopc. fragments of interest were gel-purified and ligated to yield half clone pl. quarter clones pde and pef were digested with nco i. one nco i cut was in the virus cdna insert on each bac, and the other in the sopc gene. fragments of interest were purified and ligated to yield half clone pr. half clones were digested with mlu i and pspom i. fragments of interest were gel purified and ligated into the full length clone prscv. full-length bac clones were linearized with not i, extracted with phenol-chloroform, and transcribed with the mmessage-mmachine ® t (ambion, usa) at an input of μg of dna per μl reaction. a pcr product spanning the nucleocapsid reading frame and the genomic 'prime end was generated with primers n-fwd ( '-ggccatttaggtgacactatagatgtctgataatggac-cccaatc), the underlined sequence representing an sp promoter) and frev ( '-ttttttttttttttttttttgtcat-tctcctaagaagc- '). the product was purified and transcribed with mmessage-mmachine sp kit. transcripts from both in-vitro transcription reactions were quantified photometrically. genomic transcripts and n transcripts were co-electroporated at a : ratio into bhk- cells, using a genepulser instrument (biorad, germany) with two pulses of . kv, μf and maximal resistance. cells were left at room temperature for minutes and seeded in cm flasks. in a biosafety- laboratory, electroporated bhk- cells were incubated at °c for hours. supernatants were serially diluted and transferred to vero cells. using % seaplaque ® agarose overlay (biozym, germany), three rounds of plaque purification were performed for each recombinant virus. to distinguish between the two genotypes, two different rt-pcrs were performed. rt-pcr used primers fwd ( '-cagctgcgtgcaagatcagt- ') and rev ( '-ccctagtgttgtaccttacaag- '), thus comprising orf b and yielding a bp fragment for rscv, while giving a bp fragment for r bΔtmd. for rt-pcr the identical reverse primer was used but the binding site of the sense primer fwd ( '-tagcctttctgctattc-cttgt- ') was placed in orf b, recognising the nt only present in rscv but deleted in r bΔtmd, hence a pcr product was only obtained for rscv. cloning of orf a, b and b del orf a (nts to of sars-cov genome gen-bank accession number ay ) was amplified using primers '-caccatgaaaattattctcttcctgaca- ' (fwd) and '-tcattctgtctttctcttaatggt- ' (rev), and cloned into pcdna . . because of low expression rates of the protein (data not shown) the insert was cloned into the high level expression vector pcaggs [ ] , using kpni and noti. orf b gene (nts to ) and the deletion mutant orf b del gene (nts deletion of nts ) were amplified using primers '-ctagaattcctcgagacaatgagaagtttcatgttc- ' and '-atcgtcgacctcgagtcaccattaagagaaa-gacag- ', and cloned into pi. vector (kind gift of jim robertson, national institute for biological standards and control, hertfordshire, uk) with a t promoter that was inserted by standard cloning procedures. expression of constructs was verified by coupled in-vitro transcription and translation using the tnt t coupled reticulocyte lysate system (promega, mannheim, germany) and immunofluorescence analysis of transfected cells (data not shown). transfection of cells was performed using the calciumphosphate transfection kit (invitrogen) according to the manufacturer's instructions. cells were transfected with . μg of the interferon (ifn)-stimulated response element (isre)-driven firefly luciferase reporter plasmid phisg- -luc (kind gift of d. levy, new york university school of medicine, new york), . μg of the constitutive renilla luciferase expression plasmid prl-sv (promega) and μg of the plasmid of interest. μg of herring sperm dna (promega) were transfected along with the plasmids to optimize dna uptake. h post transfection, cells were infected with sev ( hemagglutinating units) to induce the type i ifn response or were not infected. at h post infection (p.i.), cells were harvested and lysed in μl of passive lysis buffer (promega, mannheim, germany). subsequent luciferase assays were performed by using the promega dual luciferase assay system according to the manufacturer's instructions. relative renilla luciferase production was used to normalize for transfection efficiency. cells were seeded on chamber slides (μslide ® well, ibidi, martinsried, germany) and infected with sars-cov t a multiplicity of infection of . after hours cells were washed once with pbs and fixed in ice cold acetone for minutes. prior to antibody staining, cells were washed three times with pbs. reconvalescent sars patient serum from our own diagnostic laboratory was diluted : in pbs-t. rabbit polyclonal antibody against the ddddk tag (abcam, uk) was diluted : in pbs-t. cells were overlaid with μl of antibody solution, incubated at °c for hour, and washed four times for minutes with pbs containing . % tween (pbs-t). fluoresceinconjugated goat anti-human or anti-rabbit igg serum (calbiochem/vwr, darmstadt, germany) was diluted : in pbs-t and incubated at °c for minutes. cells were washed times with pbs. chambers were overlaid with μl of pbs and a few drops of mineral oil. fluorescence was analysed on an inverted fluorescence microscope. subconfluent -lp cells in six well-plates were infected, harvested at different time points after infection, and pelleted by centrifugation for minutes at rpm. pellets were dissolved in μl of × chaps buffer containing mm pmsf and mm dtt followed by three freeze/thaw cycles. nuclei were pelleted by centrifugation for minutes at , g and the clarified lysate was dissolved in × sds loading buffer. μl of postnuclear lysate were loaded on precast % bis-tris gradient gels. separated proteins were electroblotted on nitrocellulose membranes (whatman, dassel, germany) and blocked for h with × rotiblock (roth, karlsruhe, germany). membranes were washed with pbs and incubated over night with primary antibody at °c. rabbit polyclonal anti-caspase- and anti-parp antibodies (cell signaling, danvers, usa) were diluted : in pbs-t. rabbit anti-flag antibody (abcam, cambridge, uk) was diluted : in pbs-t. membranes were washed times for minutes with pbs-t. horseradish peroxidase-conjugated goat anti-rabbit antibody (cell signaling, danvers, usa) was used at : dilution in pbs-t and incubated on membranes for hour. membranes were washed four times with pbs-t before lumiglo reagent (cell signaling, danvers, usa) was added. membranes were exposed to scientific imaging film (sigma-aldrich, munich, germany) for appropriate times before development. in overexpression experiments, vero e cells were transfected with lipofectamine (invitrogen) according to the manufacturer's instructions. × cells were transfected with μg of empty pi. vector, pcaggs-orf a, pi. -orf b or pi. -orf b del, respectively. at h post transfection, cells were lysed in μl × chaps buffer. proteins were separated on % sds polyacrylamide gels, transferred onto pvdf membranes and blocked for h with % skim milk (w/v) in pbs-t. 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transfectants with a novel eukaryotic vector we are grateful to jindrich cinatl, friedemann weber, christopher basler, beate kümmerer, and jim robertson for donations of viruses or cells. we thank toni rieger for his kind help during hamster inoculations. interferon alpha was detected with the human ifn alpha elisa kit (pbl interferonsource, piscataway, usa) according to the manufacturer's instructions. briefly, μl of supernatant of samples and controls were added to pre-coated microtiter plates and incubated at room temperature for hour, followed by one washing step, addition of antibody solution and another hour of incubation. after three washing steps, μl of hrp conjugate concentrate were added and incubated for hour. the plate was washed four times and tmb substrate was added. after minutes of incubation stop solution was added and absorbance was determined at nm. total rna was prepared from -lp cells in -well plates with trizol ® reagent (invitrogen, usa) according to the manufacturer's instructions. rna was quantified photometrically and ng per reaction were analysed by realtime rt-pcr. interferon beta mrna was amplified with primers ifn fwd ( '-gaactttgacatccctgagga-gatt- ') and ifn rev ( '-ggagcatctcatagatggt-caatg- '), and '-nuclease probe ifn -p (fam-cagcagttccagaaggaggacgcc-tamra). gapdh mrna was detected in parallel with primers gapdhfwd ( '-aggtggtctcctctgacttcaaca- '), gapdhrev ( '-agtggtcgttgagggcaatg- '), and probe gapdh-p (fam-cacccactcctccacctttgacgct-tamra). reactions using the onestep rt-pcr kit (qiagen, hilden, germany) comprised °c for minutes, followed by °c for minutes and cycles of °c for seconds and °c for seconds. for both genes standard curves were generated from limiting dilution series of quantified rna. the dilution end-points were defined as one pcr unit for each gene. log pcr units for each experimental sample were calculated from the linear equations of the dilution series. interferon beta quantity was normalised to gapdh quantity by subtraction of logarithmic quantities (interferon gapdh). infections were performed with rscv and r bΔtmd. heat-inactivated rscv served as the mock-control. syrian golden hamsters (strain lvg, charles river laboratories) were infected via the intranasal route with pfu each. μl of virus solution were applied. hamsters were sacrificed at indicated days post infection. lungs were prepared in total, weighed, and homogenized. tissue was suspended to a concentration of . g/ml in complete dmem before analysis by rt-pcr or cell culture. the authors declare that they have no competing interests. sp constructed the infectious clone and conducted all experiments with recombinant viruses. vk and em designed and carried out the overexpression experiments and the reporter assays and/or critically revised the manuscript. vd conducted hamster infections. kg participated in construction of the infectious clone. cd designed the study, participated in the construction of the infectious clone, and wrote the manuscript. all authors took part in manuscript preparation. all authors read and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge key: cord- -d a y qh authors: khetawat, dimple; broder, christopher c title: a functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: d a y qh background: hendra virus (hev) and nipah virus (niv) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in queensland, australia in and peninsular malaysia in / respectively and classified within the new henipavirus genus. both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. extensive laboratory studies on the host cell infection stage of hev and niv and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level- (bsl- ) containment. to circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (gfp) gene encoding human immunodeficiency virus type- (hiv- ) genome in conjunction with the hev and niv fusion (f) and attachment (g) glycoproteins. results: functional retrovirus particles pseudotyped with henipavirus f and g glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the hev and niv receptors ephrinb or b on target cells. the functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the f or only g glycoprotein were prepared and assayed. virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting c-terminal heptad (hr- ) peptide, a well-characterized, cross-reactive, neutralizing human mab specific for the henipavirus g glycoprotein, and soluble ephrinb and b receptors. in addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of f in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of niv and hev f. conclusions: together, these results demonstrate that a specific henipavirus entry assay has been developed using niv or hev f and g glycoprotein pseudotyped reporter-gene encoding retrovirus particles. this assay can be conducted safely under bsl- conditions and will be a useful tool for measuring henipavirus entry and studying f and g glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors. hendra virus (hev) emerged in in two separate outbreaks of severe respiratory disease in horses with subsequent transmission to humans resulting from close contact with infected horses. nipah virus (niv) was later determined to be the causative agent of a major outbreak of disease in pigs in - along with cases of febrile encephalitis among people in malaysia and singapore who were in close contact exposure to infected pigs (reviewed in [ , ] ). phylogenetic analysis revealed that hev and niv are distinct members of the paramyxoviridae [ , ] and are now the prototypic members of the new genus henipavirus within the paramyxovirus family [ ] . pteropid fruit bats, commonly known as flying foxes in the family pteropodidae, are the principal natural reservoirs for both niv and hev (reviewed in [ ] ) however recent evidence of henipavirus infection in a wider range of both frugivorous and insectivorous bats has been reported [ , ] . since their identification, both hev and niv have caused repeated spillover events. there have been recognized occurrences of hev in australia since with at least one occurrence per year since , the most recent in may . every outbreak of hev has involved horses as the initial infected host, causing lethal respiratory disease and encephalitis, along with a total of seven human cases arising from exposure to infected horses, among which four have been fatal and the most recent in (reviewed in [ ] ) [ ] [ ] [ ] . by comparison there have been more than a dozen occurrences of niv emergence since its initial recognition, most appearing in bangladesh and india (reviewed [ ] ) and the most recent in march [ ] and january [ ] . among these spillover events of niv the human mortality rate has been higher (~ %) along with evidence of person-to-person transmission [ , ] and direct transmission of virus from flying foxes to humans via contaminated food [ ] . in contrast to other paramyxoviruses, niv and hev exhibit an extremely broad host tropism and in addition to bats, horses, pigs and humans, natural and/or experimental infections have also been reported in cats, dogs, guinea pigs, hamsters (reviewed in [ ] ), ferrets [ ] and some nonhuman primates, the squirrel monkey [ ] and the african green monkey [ , ] . in those hosts susceptible to henipavirus-induced pathology, the disease is characterized as a widespread multisystemic vasculitis, with virus replication and associated pathology in highly vascularized tissues including the lung, spleen and brain [ , ] . both the broad host and tissue tropisms exhibited by niv and hev can for the most part be explained by the highly conserved and broadly expressed nature of the receptors the henipaviruses employ, the ephrinb and b ligands [ ] [ ] [ ] [ ] which are members of a large family of important signaling proteins involved in cellcell interactions (reviewed in [ , ] ). niv and hev possess two envelope glycoproteins anchored within the viral membrane, a trimeric fusion (f) and a tetrameric attachment (g) glycoprotein (reviewed in [ ] ). the f glycoprotein is initially synthesized as a precursor f which is cleaved into the disulfide-linked f and f subunits by cathepsin l within the host cell [ ] . the g glycoprotein consists of a stalk domain and globular head and g monomers form disulfide-linked dimers that associate in pairs forming tetramers [ ] . the f and g oligomers associate within the membrane and g is responsible for engaging receptors, which in turn triggers f-mediated membrane fusion (reviewed in [ ] ). the f and g glycoproteins of niv and hev share~ % and % amino acid identity and both niv and hev can elicit cross-reactive anti-envelope glycoprotein antibody responses [ ] . it has also been demonstrated that f and g of niv and hev can efficiently complement each other in a heterotypic manner in cell-fusion assays [ ] . the henipavirus f and g glycoproteins share many of the general structural features found in the envelope glycoproteins of other paramyxoviruses, and recently the structure of both receptor-bound and unbound forms of the globular head domain of niv g have been reported [ , ] . because of their highly pathogenic nature and lack of approved vaccines or therapeutics, hev and niv are classified as biological safety level- (bsl- ) select agents by the centers for disease control and prevention (cdc) and as priority pathogens by the national institute of allergy and infectious diseases (niaid), having the potential to cause significant morbidity and mortality in humans and major economic and public health impacts (reviewed [ ] ). these restrictions have somewhat limited detailed studies on virus entry and their envelope glycoprotein functions in the context of a viral particle. to circumvent these restrictions, virus pseudotyping systems have been examined, where the envelope glycoproteins from one virus are incorporated into the progeny virions of another that lacks its own envelope glycoprotein(s), effectively changing the host range and tropism of the virus. for example, the f and g envelope glycoproteins of niv have been successfully incorporated into recombinant vesicular stomatitis virus (vsv) lacking vsv g glycoprotein (vsv-Δg) and encoding green fluorescent protein (gfp) [ , ] . other widely employed viral pseudotyping systems are those based on retroviral vectors, and lentiviral vectors have emerged as promising tools for a variety gene-delivery studies and can efficiently transduce proliferating as well as quiescent cells (reviewed in [ ] ). virus pseudotyping systems have been useful for the study of otherwise highly pathogenic viral agents such as ebola and marburg viruses, severe acute respiratory syndrome (sars) coronavirus (sars-cov) and influenza virus [ ] [ ] [ ] . here, building on the initial findings of kobayashi et al., [ ] , who first demonstrated that simian immunodeficiency virus from african green monkey (sivagm) could be functionally pseudotyped with the f and hemagglutinin-neuraminidase (hn) glycoproteins of sendai virus (sev), we demonstrate for the first time that the f and g envelope glycoproteins of niv and hev, a cellular protein receptor using paramyxovirus, can also be functionally pseudotyped into lentivirus particles using either a luciferase or gfp reporter gene encoding hiv- genome. these hiv- based, henipavirus glycoprotein pseudotyped particles exhibited the same cellular tropism characteristics as authentic niv and hev, and virus entry was specifically inhibited by antiviral agents that target the henipaviruses. the pseudotyped particles could be readily concentrated by ultracentrifugation without any loss of infectivity, and using this system we also examined the incorporation of f and g glycoproteins into virions, and explored the infectivity and pseudotyping efficiency of cytoplasmic tail truncated versions of f. this lentivirus-based henipavirus glycoprotein pseudotyped particle infection assay can also be conducted safely under bsl- conditions and will be a useful tool for measuring henipavirus entry and for studying f and g glycoprotein function in the context of virus particle entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors. it is often desirable to study the functions of viral envelope glycoproteins that are involved in attachment, membrane fusion and entry in the context of a viral particle. for example, infectivity experiments using virus particles can confirm observations made from cell-cell fusion assays, studies on virus tropism, or during the characterization of antiviral agents targeting various stages in the virus entry process [ ] . however, work with infectious henipaviruses is restricted to bsl- containment which raises both cost and safety issues. to counter this limitation, we sought to develop a henipavirus envelope glycoprotein pseudotyping system using reporter geneencoding lentivirus vectors, which would provide a virus entry assay based on the function of the f and g glycoproteins that could be safely and routinely carried out under bsl- conditions. to test this possibility, pseudotyped retrovirus particles were produced by transfection using pnl - -luc-e-r + , a plasmid containing the hiv- proviral clone nl - which encodes luciferase and does not produce the hiv- envelope glycoprotein [ ] along with pcaggs expression vectors encoding the niv or hev f and g glycoproteins. the preparations of henipavirus glycoprotein pseudotyped virus particles and control virus particles lacking the glycoproteins were normalized for p content by elisa (see methods) and used to infect several human cell lines, t, u , hosx t and tk -, long known to be permissive for henipavirus-mediated cell-cell fusion [ , ] and the henipavirus receptor (ephrinb and b ) negative and fusion and infection resistant cell line hela-usu [ ] . pseudotyped virus particles generated with the niv f and g glycoproteins were able to infect and produce luciferase reporter gene activity at various levels on all permissive receptor expressing cells ( figure a ) while no signal was observed with the receptor negative hela-usu or with control virus particles generated by transfection with empty vector (pcaggs). surprisingly however, virus particles produced using the pcaggs expression plasmids encoding the hev f and g glycoproteins were consistently non-functional as measured by luciferase activity (data not shown). the expression vector pcaggs is a mammalian expression vector with the cytomegalovirus (cmv) immediate early enhancer linked with the chicken β-actin promoter (cag promoter) [ ] . it has an intron with the splice acceptor site from the rabbit β-globin gene, which results in the splicing of the pre-mrna, increasing the stability of the expressed mrna and enhancing the production of an encoded protein. although these features make pcaggs an efficient vector for the expression of genes in the nucleus, we found it problematic for the expression of the hev g glycoprotein, an rna virus gene normally expressed in the cytoplasm of an infected cell, and expression levels of hev g were significantly lower in comparison to niv g in the same system (data not shown). analysis of the hev g gene cloned in pcaggs using splice site prediction software from embl-ebi http://www.ebi.ac.uk/asd-srv/wb.cgi?method= revealed possible splice sites within hev g coding region ( figure b) , while none were present in the niv g glycoprotein pcaggs construct (additional file : fig. s ). mutations were introduced by site-directed mutagenesis to remove the predicted splice sites singly or in different combinations, keeping the amino acid coding sequence unaltered, and a panel of seven (sm -sm ) hev g mutant clones were generated ( figure b) . the hev g splice mutant constructs were then tested for expression by plasmid transfection which indicated that the removal of these predicted splice sites improved hev g glycoprotein production, and removal of all three sites was optimal, and mrna expression and alternative splicing patterns were confirmed by northern blot analysis (results not shown). a series of pseudotyped virus particles were prepared using hev f along with each of hev g splice mutants (sm -sm ). in addition, control virus particles were also prepared using hev f along with empty vector (pcaggs), wild-type hev g, or wild-type niv g. this series of pseudotyped virus particles were then used to infect t target cells, and as shown in figure c , the hev g splice mutant sm ( putative splice sites removed) in combination with hev f was able to produce functional pseudotyped particles, as measured by luciferase reporter gene activity, to signal levels comparable to niv f and g bearing particles ( figure a ). the remainder of the hev g splice mutants (sm -sm ) did show low levels of reporter gene signal, whereas the wildtype hev g did not. these results demonstrate that the splice site removal by mutation in hev g-sm restores the ability of hev g to be expressed in the context of pcaggs, thus allowing its incorporation into the lentivirus particles. in addition, functional particles were also generated using hev f in heterotypic combination with niv g, confirming the previous heterotypic cell-cell fusion activities observed with the henipaviruses [ ] . the heterotypic pseudotyped particles yielded reporter gene activity essentially equivalent to the hev g-sm and hev f particles ( figure c ) and similar to the signals obtained with niv f and g bearing particles ( figure a ). to confirm these findings and demonstrate an expanded utility of the henipavirus envelope glycoprotein pseudotyping systems, niv and hev f and g glycoprotein bearing lentivirus particles were prepared with the gfp reporter gene encoding construct pnl - -gfp-e-r + and used to infect receptor positive t cells ( figure ). here, productively infected cells were visualized using a fluorescent microscope hrs post-infection and fluorescent cells were observed only in those cells infected with pseudotyped virions prepared with either niv f and niv g or hev f and hev g sm . no gfp expressing cells were observed in those wells infected with virions prepared with empty vector (pcaggs) or virus particles prepared with hev f and wild-type hev g. to examine the cellular infection specificity of the hev and niv f and g pseudotyped particles, several henipavirus specific reagents capable of blocking virus infection were tested for their ability to inhibit the infection of the henipavirus pseudotypes. virus particles were prepared as before and then mixed with various inhibitors ( figure ). the henipavirus specific peptide fusion inhibitor niv-fc , a amino acid peptide corresponding to the henipavirus heptad repeat region (hr- ) of the f glycoprotein [ , ] , completely blocked the entry of the henipavirus pseudotypes as measured by luciferase reporter gene activity. the niv-fc peptide functions in an analogous manner to the hiv- specific fusion inhibitor enfuvirtide (fuzeon™, formerly t- ) [ , ] , and specifically blocks the formation of the class fusion glycoprotein structure known as the -helix bundle of the f glycoprotein preventing f-mediated membrane fusion and subsequent virion entry. a scrambled version of the peptide (sc niv-fc ) was used as a negative control. infection specificity was also examined by inhibition with the cross-reactive anti-henipavirus g glycoprotein human monoclonal antibody (mab) m . [ , ] . the m . mab neutralizes henipaviruses by specifically binding and blocking the ephrin-b and -b receptor-binding region on the henipavirus g glycoprotein. as shown in figure , infection of either the hev or niv pseudotypes was completely blocked by mab m . confirming that their entry and resultant luciferase signal is specifically mediated by the attachment and subsequent fusion triggering functions of their henipavirus g glycoproteins. in addition, the binding and infection of the henipavirus pseudotypes to target cells could be blocked by recombinant, soluble ephrin-b and -b receptors ( figure ). hev and niv f and g bearing particles pre-incubated with soluble ephrin-b or -b were unable to infect host cells as was previously shown with infectious virus [ ] . also, in a reciprocal manner, recombinant soluble niv g (sg) could block entry of either henipavirus pseudotype as was similar to earlier observations made with hev sg in infectious virus entry. together, these results demonstrate the specificity of the henipavirus f and g glycoprotein bearing pseudotyped virus entry assay and its potential utility in screening specific henipavirus entry inhibitors. previous studies have demonstrated that efficient incorporation of heterologous envelope glycoproteins into hiv- or murine leukemia virus (mlv) particles often depended on the removal of part or all of the cytoplasmic tail domains from the pseudotyping glycoproteins [ , [ ] [ ] [ ] . to explore whether a similar feature was occurring in the henipavirus pseudotyping system here, a series of seven cytoplasmic tail truncation mutations in each henipavirus f glycoprotein were generated, designated fΔct to fΔct , by introducing translational stop codons into the coding sequence of the niv and hev f gene ( figure ) . the fΔct and fΔct constructs of both the niv and hev f, differ by only one additional deleted valine residue to better ensure complete removal of the cytoplasmic tail domain. because cytoplasmic tail truncations of membrane anchored proteins could affect proper folding and transport, we first examined the levels of cell surface expressed f and compared the series of truncated mutant f constructs to each wild-type f, using a cell surface biotinylation assay [ ] . the series of niv and hev f glycoprotein mutants and each wild-type f were expressed by plasmid transfection in hela-usu cells, both in the presence and absence of their homologous g glycoprotein partner, and surface proteins were biotin labeled, precipitated with avidin-agarose, and analyzed by western blot assay using an f specific antisera ( figure ). the wild-type niv f precursor was cleaved and detected. fΔct , fΔct appeared less efficiently cleaved (levels of f versus f ) as compared to wild-type niv f. a significant amount of each of the niv fΔct , fΔct , fΔct , fΔct and fΔct constructs were cleaved, with the fΔct appearing highly processed although its overall expression was lower in comparison to others. the ratio of cleaved to uncleaved f (f to f ) on the cell surface was approximately equal ( : ) when the complete retention and endocytosis motif (ysrl) [ , ] was retained, beginning with the fΔct constructs. notably, the coexpression of niv g did not appear to significantly alter the expression and cleavage patterns of niv f ( figure ). the retention of amino acid residues from the endocytosis motif ysrl to residues edrrv in the cytoplasmic tail appeared to allow for more efficient f processing, as evidenced by the greater levels of f observed with these niv constructs (niv fΔct to fΔct ) ( figure ) in comparison to niv fΔct , fΔct and fΔct which lack the ysrl motif. in addition, the cell surface levels of f (primarily f ) observed with the fΔct , fΔct and fΔct constructs appeared greater in comparison to the fΔct , fΔct , fΔct and fΔct constructs, and this mostly likely reflects the reduced ability of the f precursor to be endocytosed and processed by cathepsin l [ , ] . similar results were obtained when the series of hev f cytoplasmic tail truncation mutants were examined in parallel, and the hev f constructs fΔct , fΔct and fΔct revealed greater cell surface expression levels of f with less efficient processing as measured by the detection of f , whereas the hev f constructs, fΔct through fΔct revealed greater f precursor processing but perhaps an overall lower level of expression ( figure ) . a variable and doublet appearance of hev f has been observed previously [ , , ] . as with the niv f truncation mutants the coexpression of the hev f panel along with their hev g glycoprotein partner did not significantly alter the hev f expression and cleavage patterns observed in cell surface biotinylation assays. having characterized the expression and processing of the cytoplasmic tail truncation mutants of both niv and hev f glycoprotein, we next examined their biological function in cell-cell membrane fusion assays. membrane fusion was assessed using the well-characterized vaccinia virus-based, reporter-gene, cell-cell fusion assay [ ] . this assay has also been used extensively in earlier reports on the characterization of hev and nivmediated membrane fusion and tropism [ , , ] . the series of f glycoprotein truncation mutants for both hev and niv were expressed, along with their respective partner g glycoprotein, in hela-usu cells (effector cells) and cell-cell fusion reactions were carried out using target cells of either receptor negative hela-usu (control) or fusion permissive t cells, and results are shown in figure . for niv f, the removal of most of the cytoplasmic tail domain from f (fΔct and fΔct ), which also reduced f processing, impaired their fusogenic potential as would be expected, whereas the fusogenic activity of niv fΔct , fΔct , fΔct , fΔct and fΔct were either equivalent or slightly elevated in comparison to wild-type niv f. the cell-cell fusion assay with the series of hev f truncation mutants generated slightly more variable results in contrast to niv f, though all possessed some fusogenic activity. in general there was only a slight reduction in fusion with hev f, fΔct and fΔct , while fΔct , fΔct and fΔct were essentially equivalent to wild-type hev f, while lower fusion signals were seen with hev fΔct and fΔct , which could be related to an overall lower expression level as seen in figure . a comparison of the results in figure and figure suggests that niv f processing appears to correlate with cell-cell fusion signals; whereas cell-cell fusion activity was readily apparent in several hev f truncation mutants possessing a markedly lower level of f processing, however these are independent experiments and a direct comparison may be miss-leading. figure cell surface expression of truncation mutants of the henipavirus f glycoprotein. the various f cytoplasmic tail truncation mutants alone or together with their g glycoprotein partner were transfected into hela-usu cells. at hr post transfection, cell surface proteins were biotinylated and precipitated with avidin agarose beads, and the precipitated proteins were processed for western blot analysis as detailed in the methods and probed using the anti f specific antisera. this experiment was performed twice and representative experiment is shown in the figure. khetawat and broder virology journal , : http://www.virologyj.com/content/ / / we next examined the efficiency of the various cytoplasmic tail truncation mutants of the niv and hev f glycoproteins to be incorporated into lentivirus-based pseudotypes. pseudotyped lentivirus particles were prepared as before using the series of cytoplasmic tail truncation mutants along with their partner g glycoprotein. three types of control virus particles were also prepared using either empty vector (pcaggs) or each species of wild-type f glycoprotein alone or each species of g glycoprotein alone. pseudotyped virus particle preparations were filtered, purified by centrifugation through a sucrose cushion, normalized for p content by elisa and used to infect t target cells. following infection and incubation for h, cells were processed and luciferase activity was measured. as shown in figure a and b, pseudovirus particles prepared using the truncation mutants fΔct , fΔct , and fΔct f glycoproteins produced significantly greater levels of luciferase activity as compared to virus particles made with wild-type f. to evaluate whether the differences in infectivity, as measured by luciferase reporter gene activity, by the various pseudovirus types correlated to the extent of incorporation of the mutant f glycoproteins into lentivirus particles, equal amounts of virus particles based on p content were lysed and analyzed by western blot. this analysis revealed that incorporation of f into either the niv or hev pseudotyped virions was greater with the fΔct , fΔct and fΔct constructs, and that f was the predominant species present in the virions ( figure c ). these results together with cell surface expression pattern of the niv and hev wild-type and truncation mutants demonstrate that, in general, the amount of incorporation of the f glycoproteins in the pseudotyped particles appears to correlate well with the level of expression of these proteins on the surface of the producer cells. this was also true in the amount of wild-type niv or hev f alone bearing particles which can be noted when comparing figure and figure c . removal of the endocytosis motif from the fusion protein prevents its transportation to the endosome and subsequent cleavage of f into f and f by cathepsin l, which explains the predominance of f in the fΔct , fΔct and fΔct constructs which lack the endocytosis motif ysrl. interestingly, in both the niv and hev f glycoprotein mutant series, the higher infectivity of the fΔct , fΔct and fΔct bearing pseudotypes in comparison to wild-type was notable, and might be attributed to the greater levels of incorporation of these f glycoproteins into the particles, except in the case of wild-type niv f and g bearing particles and the reason for this later observation is unclear at present. alternatively however, and also of interest is that the high infectivity signal and predominance of f in the pseudotypes prepared with fΔct , fΔct and fΔct could argue for a role of endocytosis followed by cathepsin l processing of f and subsequent productive fusion and infection. in the present study we have detailed a new and readily adaptable, reporter-gene containing, lentivirus-based pseudotyping system which utilizes functional f and g envelope glycoproteins of the henipaviruses; niv and hev. importantly, like other virus envelope glycoprotein pseudotyping systems, this assay can be conducted safely under bsl- , a condition which is relevant considering the otherwise highly pathogenic nature of infectious niv and hev. we also demonstrate, by several measures, that khetawat and broder virology journal , : http://www.virologyj.com/content/ / / this henipavirus pseudotyping system faithfully recapitulated the natural niv or hev cell attachment and viral glycoprotein-mediated membrane fusion stages of infection. the henipaviruses bind and infect their host cells by a specific attachment step to the cell surface expressed proteins ephrin-b and -b [ ] [ ] [ ] [ ] . the current and widely accepted model of paramyxovirus mediated membrane fusion postulates that upon receptor binding the viral attachment glycoprotein triggers conformational changes in the f glycoprotein, a class i viral fusion glycoprotein. the receptor-induced triggering event is presumed to involve direct contacts between an attachment and fusion glycoprotein and this activation process facilitates a series of conformational changes in f and the glycoprotein transitions into its post-fusion, six-helix-bundle conformation concomitant with the merging of the viral membrane envelope and the host cell plasma membrane [ , ] . however, all of the details of the entire receptor binding and fusion activation process have yet to be defined. an important feature of many class i fusion glycoproteins is the two α-helical regions referred to as heptad repeat (hr) domains that are involved in the formation of the sixhelix-bundle structure [ , ] . hr- is located proximal to the amino (n)-terminal fusion peptide and hr- precedes the transmembrane domain near the carboxyl (c)terminus. peptide sequences from either hr domain of the f glycoprotein of several paramyxoviruses, including hev and niv, have been shown to be inhibitors of the f-mediated membrane fusion step in both cell-cell fusion and virus infection assays [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] . here, as has been shown with infectious virus or cellcell fusion assays, the infection by niv and hev f and g lentivirus pseudotypes was completely blocked by the hr- based fusion inhibiting peptide (niv-fc ) [ ] . a number of other tests were also conducted to demonstrate the specificity of the henipavirus pseudotyping system in addition to using the henipavirus peptide fusion inhibitors. in competition assays, the infection of the pseudotypes could also be specifically blocked using recombinant, soluble ephrin-b or ephrin-b receptor proteins as was previously shown with both henipavirus-mediated membrane fusion as well as live virus infection assays [ ] . in a similar fashion, recombinant, soluble henipavirus g glycoprotein (sg) was also able to completely inhibit the infection of either hev or niv pseudotypes by blocking receptor binding, which had been demonstrated previously in both henipavirus-mediated membrane fusion and live virus infection assays [ ] . finally, the infection by the niv and hev pseudotypes could also be completely blocked using a well-characterized, cross-reactive human mab (m . ) that is specific for the henipavirus g glycoprotein [ , ] . thus, by a wide variety of wellknown and well-characterized approaches the functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system developed here, accurately recapitulates the receptor binding, membrane fusion and infection stages of live hev and niv. because of both the highly pathogenic features of niv and hev, which restricts the use of infectious virus to bsl- containment, and the labor intensive nature and challenges associated with a reverse genetics approach, extensive and detailed structural and functional studies on the henipavirus envelope glycoproteins in the context of a viral particle has been limited. to demonstrate the utility of the henipavirus pseudotyping system here, we generated and tested an extensive panel of cytoplasmic tail domain truncation mutants of the niv and hev f glycoprotein, and examined the influence of this domain of f on its ability to be incorporated into this budding particles as well as its fusion activity in the context of a viral particle. here, it was observed that the deletion of essentially the entire f cytoplasmic tail domain, most notably with the niv f glycoprotein and to a lesser degree with that of hev f, impaired their fusogenic activity in the context of a cell-cell fusion assay. these findings were in contrast with previous observations made on the envelope glycoproteins of certain lentiviruses. studies with human immunodeficiency virus type (hiv- ) and simian immunodeficiency virus (siv) envelope (env) glycoproteins have shown that cytoplasmic domain truncation mutants exhibit significantly enhanced env fusogenic activity as measured by syncytium formation [ , ] . in addition, studies with murine leukemia virus have demonstrated that naturally occurring late cleavage of a small carboxy terminal sequence, designated as the r peptide or p e, in the cytoplasmic tail results in considerably enhanced cell-to-cell fusion activity [ , ] . whereas for a paramyxovirus f glycoprotein, cytoplasmic tail deletions in simian virus (sv ) [ ] , newcastle disease virus [ ] , and human parainfluenza virus (hpiv) type (hpiv- ) revealed significantly reduced syncytium formation, except in one example with hpiv- , where similar deletions did not affect membrane fusion [ ] . overall, with the exception of the results with hpiv- , these studies also demonstrated that subsequent additions of parts of the deleted cytoplasmic tail sequences restored the fusogenic potential of those f glycoproteins. in the case of henipaviruses, one explanation to account for the reduced fusion activity of the entire cytoplasmic tail deleted constructs is poor endocytosis and subsequent cathepsin l processing of f and the analysis of the surface expressed levels of niv f versus f in the cytoplasmic tail domain truncation mutants support this conclusion, but to a lesser extent with that of the hev f truncation mutants. however, although the cell-cell fusogenic results with the truncation constructs of the henipavirus f glycoproteins reported here were similar to the majority of the observations made with other paramyxoviruses, whether as a result of f precursor processing or by some other mechanism, the cytoplasmic tail deleted hev and niv f glycoproteins in the context of the virus particle pseudotyping system, revealed an opposing result. in general, the higher levels of pseudotyped particle infectivity signal correlated with an overall greater level of incorporated f glycoprotein. interestingly however, the highest luciferase signals in the virus infection assays also correlated with a greater level of unprocessed f in the particles, particularly with fΔct , fΔct and fΔct in which most of the cytoplasmic tail was deleted. potentially, the greater luciferase signals in these instances (fΔct , fΔct and fΔct ) could be due to particle endocytosis following receptor binding [ ] and subsequent f processing by cathepsin l [ ] . the pseudotyping system described here offers one system, albeit artificial, to explore the possibility of a productive early endocytic route of henipavirus infection. taken together, this henipavirus pseudotyping system shown here offers a useful tool for measuring not only henipavirus entry and assaying and characterizing virus neutralizing antibodies and virus entry inhibitors, but also offers a highly versatile platform for studying f and g glycoprotein function in the context of a virus particle during infection, and one that can readily assay numerous variations or mutants of either or both the f and g henipavirus glycoproteins. functional henipavirus envelope glycoprotein pseudotyped, reporter gene encoding, lentivirus particles could be readily produced, concentrated by ultracentrifugation and stored frozen without loss of infectivity. these henipavirus pseudotyped particles maintained the same cellular tropism characteristics as authentic niv and hev, and infection of host cells by these particles could be specifically inhibited by various antiviral agents that target the henipaviruses. this henipavirus glycoprotein pseudotyped virus infection assay can be conducted safely under bsl- conditions and its utility in analyzing the viral glycoprotein function, of otherwise bsl- restricted agents, in the context of a virus particle was demonstrated in the characterization of cytoplasmic tail truncated versions of the f glycoprotein. this new henipavirus pseudotyping system will be a useful tool for measuring hev and niv entry and studying their f and g glycoprotein function in the context of virus particle, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors. u and hutk - b were obtained from the american type culture collection (atcc). recombinant human osteosarcoma cells bearing cd and cxcr (host x ) were obtained from the nih aids research and reference reagent program [ ] . the t cells were obtained from dr. g. quinnan (uniformed services university). hela-usu cell line has been described previously [ ] . hela-usu, u , host x and t cells were maintained in dulbecco's modified eagle's medium (quality biologicals, gaithersburg, md) supplemented with % cosmic calf serum (ccs) (hyclone, logan, ut) and mm l-glutamine (dmem- ). all cell cultures were maintained at °c in a humidified % co atmosphere. the hev and niv f and g envelope glycoproteins were transiently expressed using the mammalian expression vector pcaggs which contains the cag promoter and is composed of the cytomegalovirus immediate early enhancer and the chicken b-actin promoter [ ] . the hiv- pnl - -luc-e-r + or pnl - -gfp-e-r + backbone plasmids encoding the luciferase (luc) [ ] or green fluorescence protein (gfp) reporter gene were provided by dr. r. doms (university of pennsylvania). the henipavirus g and f glycoproteins were detected with a cross-reactive polyclonal mouse antiserum raised against recombinant, soluble hev g [ , ] or a rabbit polyclonal henipavirus f -specific antiserum provided by dr. l-f. wang (australian animal health laboratory, geelong, australia) respectively. the human monoclonal antibody (mab) m . igg used for inhibition of virus entry [ , , ] was provided by dr. d. dimitrov (national cancer institute-frederick, national institutes of health). the fusion inhibiting peptide niv-fc corresponding to the hr region of niv f and the nonfusion inhibiting scrambled control peptide sc-niv-fc have been previously described [ ] . recombinant, soluble ephrin-b and -b were from r&d systems, minneapolis, mn. recombinant, soluble niv g (niv sg) has been previously described [ ] fusion (f) glycoprotein constructs and mutagenesis full-length cdna clones of the niv and hev f glycoprotein genes [ , ] each including the kozak consensus sequence (ccacc) appended upstream of the initial atg [ ] were subcloned into pcaggs, generating the niv f-pcaggs and hev f-pcaggs expression vectors. the cytoplasmic tail domain truncation mutants of niv and hev f were generated by introducing stop codons corresponding to amino acid positions , , , , , and of the full-length f glycoprotein by standard pcr techniques. the niv f-pcaggs and hev f-pcaggs plasmids were used as templates, and the ' primer included an external ecori site, the kozak consensus sequence (ccacc) and f-specific sequence. the various ' primers included f specific sequence, a stop codon at the desired location and an external kpni site. all pcr products were gel purified and cloned into the topo vector (invitrogen) and subsequently subcloned into pcaggs, generating constructs niv fΔct through fΔct and hev fΔct through fΔct (figure aand b ). all constructs were sequenced confirmed. full-length cdna clones of the niv and hev g glycoprotein genes [ , ] each including the kozak consensus sequence (ccacc) appended upstream of the initial atg [ ] were subcloned into pcaggs, generating the niv g-pcaggs and hev g-pcaggs expression vectors. the splice site mutations ( figure c ) of the hev g gene were generated by site-directed mutagenesis using the quickchange ii site-directed mutagenesis kit and quickchange multi site-directed mutagenesis kit (stratagene, cedar creek, tx). the template for the mutagenesis reactions was a hev g clone in the topo plasmid (invitrogen corp., carlsbad, ca). for the first donor splice site d at nucleotide position , the ttg codon for leucine was changed to ctt. the other redundant codons for leucine are ctg, cta, ctc, and tta, but these were not effective in removing the predicted splice site. for the second donor splice site d at nucleotide position , the agt codon for serine was changed to tcg. for the third donor splice site d at nucleotide position , the ggg codon for glycine was changed to ggt. all pcr products were gel purified and cloned into topo and subsequently subcloned into pcaggs. all mutation-containing constructs were sequence verified. hela-usu cells grown in t cm flasks were transfected with the pcaggs expression constructs of niv and hev f alone or along with their partner g glycoprotein constructs with fugene reagent (roche diagnostics corp, in) for hrs. following expression, cells were rinsed three times with ice-cold phosphate buffered saline (pbs) and cell surface proteins were biotinylated using . mg/ml ez-link nhs-biotin (pierce, rockford, il) in pbs for min at °c [ ] . the reaction was quenched by washing the cell monolayer three times with ice-cold pbs before harvesting cells and preparation of cell lysates. cells were lysed in mm tris-hcl (ph . ), mm nacl, % triton x- and protease inhibitor at °c for min and one-half of each lysate was incubated with μl of % vol/vol solution of agarose-avidin d beads (vector laboratories, inc., burlingame, ca) in ip buffer ( . m nacl, . m tris, and . % triton) at °c and rotated overnight. beads were washed twice with lysis buffer followed by one wash with doc buffer ( mm tris-hcl (ph . ), mm nacl, . % sodium deoxycholate, and . % sds). samples were boiled in sds-page sample buffer with -mercaptoethanol, separated on a - % tris-glycine gradient gel (invitrogen), transferred to nitrocellulose, and probed with a cross-reactive polyclonal mouse antiserum to hev g at a concentration of : , or a rabbit polyclonal f specific antiserum at a concentration of : , . henipavirus f and g mediated fusion activities were measured using a previously described quantitative viral glycoprotein-mediated cell-cell fusion assay [ , , ] . briefly, one cell population (effector cells) is infected with a recombinant vaccinia virus expressing the t polymerase (vtf . ) and the other cell population (target cells) is infected with a vaccinia virus encoding the e. coli lacz gene (β-gal) gene under control of the t promoter (vcb r). cell-cell fusion between effector and target cell results in β-gal synthesis which can be measured by specific synthetic substrate cleavage. plasmids encoding niv or hev g along with their respective wild-type f glycoprotein partner or the various truncation mutants of f were transfected into hela-usu cells and allowed to express overnight (effector cell populations). effector cell populations were also prepared using empty vector, pcaggs, or niv or hev f glycoprotein alone as additional negative controls. the various effector cell populations were infected with vtf . and a fusion permissive t target cell population was prepared by infection with vcb r. vaccinia virus infections were carried out with a multiplicity of infection of , suspended in media and incubated at °c overnight as previously described [ , , ] . cell fusion reactions were conducted with the various cell mixtures in -well plates at °c with a ratio of envelope glycoprotein-expressing cells to target cells of : using × total cells per well in a total volume of . ml per well for . h. for quantitative analyses, nonidet p- was added ( . % vol/vol) and aliquots of the cellcell fusion lysates were assayed for β-gal at ambient temperature with the substrate chlorophenol red-dgalactopyranoside (roche diagnostics corp, in, usa). assays were performed in triplicate, and fusion results were calculated and expressed as rates of β-gal activity (change in optical density at nm per minute × , ) in an mrx microplate reader (dynatech laboratories, chantilly, va). pseudotyped, hiv- reporter gene encoding virus stocks were prepared by transfecting t cells with the reporter gene-encoding backbone plasmids pnl - -luc-e-r + or pnl - -gfp-e-r + along with the henipavirus envelope glycoprotein encoding pcaggs vectors. t cells ( . × ) were seeded in a -well flat-bottom collagen i-coated microplate (bd biosciences, durham, nc) and transfected with the expression plasmids using the fugene reagent (roche diagnostics corp, in). the dnas of the pnl - -luc-e-r + or pnl - -gfp-e-r + along with the hev or niv f and g encoding pcaggs plasmids were mixed in the ratio of : . : . , respectively, and added to μl of serum free dmem and μl fugene. the mixture was incubated at room temperature for min and then applied to the culture of t cells. after to hr incubation at °c, transfected cells were washed extensively with dmem and incubated for additional - hr with ml of dmem- , at °c in % co . the supernatants from virus particle producing cultures were then collected and were clarified by centrifugation for min at rpm, filtered through low protein binding . μm syringe filter (millipore, bedford, ma) and partially purified through % wt/vol sucrose in hepes-nacl buffer by centrifugation at × g at °c for . hr. the pellet was resuspended overnight at °c in % sucrose in hepes-nacl buffer and used immediately or stored at - °c. to measure the incorporation of the henipavirus f and g glycoproteins into pseudotyped hiv- particles, sucrose cushion purified particles were lysed in buffer containing mm tris-hcl (ph . ), mm nacl, % triton x- and protease inhibitors at °c for min. samples were boiled in sds-page sample buffer with -mercaptoethanol and separated on a - % tris-glycine gradient gels (invitrogen), transferred to nitrocellulose, and probed with a cross-reactive polyclonal mouse antiserum to hev g at a concentration of : , or a rabbit polyclonal f specific antiserum at a concentration of : , . receptor positive cell lines, seeded into -well plates at a concentration of cells per well, were infected (transduced) with pseudovirus, normalized for p antigen content using the hiv- p eia kit from beckman-coulter, and all infection experiments were carried out in triplicate wells. no deae-dextran or polybrene was used to facilitate fusion/infection by the pseudovirions. after infecting for . - hr, the cells were washed and incubated for additional - hr. for luciferase encoding particles, cells were lysed with . % triton x- in pbs and a μl aliquot of the lysate was assayed for luciferase activity using luciferase substrate (promega, madison, wi) on a mikrowin luminometer (berthold technologies model: centro lb ). for the gfp-encoding particles, the efficiency of infection was evaluated by counting the number of green cells h post-infection using olympus ix fluorescent microscope. for inhibition of pseudotyped virus infection assays, pnl - -luc-e-r + based virus particles pseudotyped with full-length niv or hev f and g envelope glycoproteins were pre-incubated with μg each of nivsg, mab . igg, niv-fc (fusion inhibiting) or sc-niv-fc (scrambled control peptide), recombinant soluble murine ephrin-b (rmefn-b /fc) or recombinant soluble human ephrin-b (rhefn-b /fc) at °c for hr. receptor positive t cells, seeded into -well plates ( cells per well) were then infected with the various pre-treated pseudotyped virus particles and processed as above. all infection experiments were performed in triplicate. additional file : splice site prediction in the pre-mrna derived from the hev g gene as cloned in pcaggs. embl-ebi splice site prediction software http://www.ebi.ac.uk/asd-srv/wb.cgi?method= was used to check for the presence of splice donor sites in the g glycoprotein constructs cloned in 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acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the views expressed in the manuscript are solely those of the authors, and they do not represent official views or opinions of the department of defense or the uniformed services university of the health science. this work was supported by nih grant ai to c.c.b. portions of this work were originally presented at the keystone symposia: cell biology of virus entry, santa fe, new mexico. authors' contributions dk contributed to the development of the henipavirus pseudovirus assay, designed and constructed all the expression constructs and carried out the experiments, identified the alternative splicing process in the original hendra virus g encoding pcaggs plasmid clone, interpreted data, and wrote the first drafts of the figures and manuscript. ccb conceived of and contributed to the development of the henipavirus pseudovirus assay provided overall supervision and financial support and wrote and prepared the final versions of the figures and manuscript. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -obvm en authors: ivancic-jelecki, jelena; slovic, anamarija; ljubin-sternak, sunčanica; mlinarić galinović, gordana; forcic, dubravko title: variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: obvm en background: small hydrophobic (sh) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (hrsv). its coding region constitutes less than % of the complete gene length, enabling sh gene to be highly variable and the sh protein highly conserved. in standard hrsv molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (hvr ) are analyzed. to what extent do the strains identical in hvr differ elsewhere in genomes is rarely investigated. our goal was to investigate whether diversity and inter-genotype differences observed for hvr are also present in the sh gene. methods: we sequenced clinical samples collected within a limited area and time frame. in this hrsv collection, rapid and significant changes in hvr occurred. results: over % of strains from this pool (containing hrsv genotypes na , on , ga , ba and ba ) would be incorrectly assumed to be identical to another strain if only the hvr region was analysed. the majority of differences found in sh gene were located in the ′ untranslated region (utr). seven indels were detected, one was genotype ga specific. an in-frame deletion of nucleotides (coding for amino acids – ) was observed in one of group a strains. fifteen different sh protein sequences were detected; % of strains possessed the consensus sequence and most of others differed from the consensus in only one amino acid (only strains differed in amino acids). the majority of differing amino acids in group a viruses had the same identity as the corresponding amino acids in group b strains. when analysis was restricted to strains with identical hvr nucleotide sequences and differing sh protein sequences, % of differences observed in the sh ectodomain were located within region coding for amino acids – . conclusions: basing hrsv molecular epidemiology studies solely on hvr largely underestimates the complexity of circulating virus populations. in strain identification, broadening of the genomic target sequence to sh gene would provide a more comprehensive insight into viral pool versatility and its evolutionary processes. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. human respiratory syncytial virus (hrsv) belongs to the orthopneumovirus genus of the pnemoviridae family [ ] and has non-segmented rna of negative polarity as its genome. the genome is approx. , nucleotides (nts) long, comprising genes that encode for proteins. the hrsv strains are divided into groups a and b based on genetic and antigenic differences [ ] . there are three proteins on the surface of mature hrsv virions: glycoprotein (g), fusion protein (f) and small hydrophobic (sh) protein. sh protein is present at very low amounts [ , ] . g and f are important for attachment and fusion with the target cell, respectively [ , ] and they can elicit the production of neutralizing antibodies [ , ] . sh protein is a short transmembrane protein (typically amino acids for group a and for group b) that is anchored by a hydrophobic signal-anchor sequence near the n-terminus, with the c-terminus oriented extracellularly. the sh ectodomain is only weakly immunogenic [ , ] . in infected cells, sh protein accumulates mostly in lipid rafts of the golgi and of the endoplasmic reticulum [ ] . it is a viroporin, forming a transmembrane pentameric ring that functions as a channel for cations and small molecules [ , ] . hrsv sh protein blocks or delays apoptosis through inhibition of the tnf-α signalling pathway [ ] but the mechanism is still not fully elucidated. in molecular epidemiology studies, hrsv surveillance and genotyping are based on sequences of the second, c-terminal hypervariable region of the g gene (hvr ). in frame duplications of and nts have occurred within this region, leading to emergence of group a strains belonging to genotype on (which originated from na , and was firstly detected in [ ] ) and of group b strains belonging to ba genotypes, respectively. to what extent do the strains identical in hvr differ elsewhere in their genomes is rarely investigated. in the analysis by agoti et al. such strains differed by at least and up to nts across the complete genome [ ] . next to hvr , the most diverse hrsv genomic region is the sh gene [ ] . unlike all other hrsv genes, the sh coding region constitutes less than % of the complete gene length (the coding regions of other hrsv genes constitute ca. - % of the gene length). following our previous analysis of hvr sequences of strains detected within a limited geographical area (the zagreb region) and limited time frame (march to march ) [ ] , the goal of this study was to investigate whether virus variability and inter-genotype differences observed for hvr are also present in the sh gene. within this pool of viruses, rapid and significant genetic changes have occurred in hvr [ ] . the subset of strains analysed in this research belong to hrsv a (na , on and ga ) and hrsv b genotypes (ba and ba ) and it included all the genotypes that were detected in zagreb, / - / . these are also the most common hrsv genotypes detected worldwide in the last decade. nasopharyngeal secretions were obtained from children who were in-patients with acute respiratory infections, hospitalized mostly due to bronchiolitis or pneumonia [ ] . based on the availability of the material, samples with previously determined sequences of the hvr region and genotype [ ] were chosen for sh gene analysis. sequences were obtained from samples. total rna was extracted from μl of clinical samples according to the method reported by chomczynski and mackey [ ] . reverse transcription was performed at °c for min, in a reaction mix containing μl of isolated rna, × pcr buffer (ge healthcare, uk), . mm of each dntp, u of rnase inhibitor (thermo fisher scientific, usa), . mm mgcl , . mm of random hexanucleotide primers and u of mulv reverse transcriptase (thermo fisher scientific, usa) in a final volume of μl. nested pcr was carried out with two sets of primers. for the first amplification, forward sh ( ′ caca gtkactgacaayaaaggagc ′) and reverse f ( ′ gttatgacactggtataccaacc ′) primers were used, followed by the second amplification with the sh ( ′ cagatcatcccaagtcatt ′) and sh ( ′ tgattgagagtgtcccaggt ′) primer pair for hrsv a strains and the sh ( ′ agccattgtctgcc agayctagag ′) and sh primer pair for hrsv b strains. ten microliters of the reverse transcription reaction mix was used for the first amplification, whereas μl of the first amplification mixture was used for the second pcr. pcr reaction mixtures contained × pcr buffer (ge healthcare, uk), μm of each dntp, . mm mgcl , . μm of each primer and u of taq polymerase (ge healthcare, uk). pcr conditions for the first amplification were: °c for min, cycles of °c/ s, °c/ s, °c/ min, followed by final extension at °c for min. the second pcr was performed under the same conditions, except the primer annealing temperature was °c, and the extension step was shortened to min. the amplification products of ca. nts were separated on a . % agarose gel. sequencing reactions were set up with gel purified dna, one of the specific primers used in the second pcr and a bigdye terminator v . cycle sequencing kit (thermo fisher scientific, usa) according to the manufacturer's protocol. sequencing and sequence analysis were performed on a genetic analyzer (thermo fisher scientific, usa). obtained sequences were submitted to national center for biotechnology information (ncbi) database, under acc. nos. mf -mf (hrsv a) and mf -mf (hrsv b). the hrsv genomic region used in the analyses (referred to as shseg) spanned bases - of strain a (acc. no. m ) and - of b (acc. no. af ), which are the prototype strains of group a and b, respectively. hvr sequences included in this study are a subset of sequences determined during our previous study on hrsv molecular epidemiology [ ] . hvr region spanned bases - of strain a and - of ba / b. the length of hvr is or nts for group a and or nts for group b. all available hrsv sequences that possessed aaaa aagtca at the end of sh ′ untranslated region (utr) and beginning of sh-g intergenic region were downloaded from ncbi database. besides the strains sequenced in this study, sequences were found, acc. nos. kf , kj , kj , kj , kj , kj , ku , ku , ku , ku , ku , ku , ku and kx . sequences were aligned using bioedit, version . . and clustalx . . sequence conservation (defined as the percentage of genomic positions identical in all strains; gaps were ignored during computing) and mean p-distance (proportion of nucleotide sites at which two sequences being compared are different) were calculated using mega software v . . the ratio of the number of non-synonymous substitutions per non-synonymous site and synonymous substitutions per synonymous site (dn/ds) was calculated with the single-likelihood ancestor counting method (slac) available on the datamonkey server (http:// www.datamonkey.org/). phylogenetic trees were generated using the maximum likelihood method with mega software v . , under the most appropriate model of nucleotide substitution determined with jmodeltest v . . . bootstrap probabilities for iterations were calculated to evaluate confidence estimates. values higher than % were considered significant. codon-based analysis of selective pressure for sh gene sequences was performed using the hyphy package available on the datamonkey server. four different methods were used: slac, fixed-effects likelihood, internal branch fixed-effects likelihood and mixed effects model of evolution. sites were considered positively or negatively selected if (a) they met the cut-off criteria of p-value of < . ; or (b) they were recognized by at least two methods. netoglyc . server (http://www.cbs.dtu.dk/services/ netoglyc/) and netnglyc . servers (http://www.cbs. dtu.dk/services/netnglyc/) were used for o-and n-glycosylation site prediction, respectively. the shseg spanned the entire sh gene (coding, ' utr and ' utr), sh-g intergenic region and g gene ' utr ( fig. ) . we obtained sequences of hrsv a strains; , and were na , on and ga strains, respectively. seventy-eight sequences belonged to hrsv b group; belonged to genotype ba and to ba . the nucleotide length of the consensus sequence was nts for group a and nts for group b strains. seven indels were detected (fig. ). in group a deletions were at positions , - , in poly a tail ( - ) and at positions - in reference to the consensus sequence a ; insertions were an additional adenosine in poly a tail and a guanosine after nucleotide ( fig. ). in group b, a deletion of an adenosine in poly a tail was observed (position - in reference to the consensus sequence b ). five indels were found only within a single genotype, but in very low percentages. deletion of nts at the beginning of the sh-g intergenic region in ga strains, preceded by adenosines was found in % of our ga strains and only in ga genotype (and therefore could be classified as genotype specific). our viral pool contained only ga strains, identical in both hvr and shseg and all collected in february . therefore we searched for other hrsv sequences that possess aaaa aagtca at the end of sh ' utr and beginning of sh-g intergenic region in ncbi database. fourteen sequences were retrieved and all belong to genotype ga (additional file : figure s ). we analysed if viruses with identical hvr sequences (considered to be identical viruses in standard molecular evolutionary analyses) had differences in shseg. some strains identical at hvr differed at shseg, and vice versa, strains different at hvr were identical at shseg (table , additional file : table s ). in our hrsv pool, if strains were inferred as being the same based only on hvr sequence analysis, strains ( . %) would have been falsely characterized. as expected, none of shseg sequences found among hrsv a strains was detected within hrsv b pool, but there was also no overlapping between the genotypes. i. e., none of the shseg sequences present in any of the hrsv genotypes included in the study were detected in strains belonging to another genotype. the phylogenetic analysis based on shseg (fig. ) did not discriminate na and on genotypes. na and on strains were placed within a common clade, separated from ga strains and from group b lineage. in group b, a single ba strain is positioned among the ba strains (fig. ) . we calculated the percentage of conservation and mean p-distance within complete hvr , complete shseg and within each individual region of the shseg (table ) . compared to hvr , shseg showed higher sequence conservation in all genotypes. difference in variability was especially evident when solely the coding region of the sh gene was compared to hvr (hvr consists of . % of coding region (for on ), . % or . % (for na ) and . % or . % (for ba )). the evolutionarily newest genotype on showed the highest conservation percentage and the lowest p-distance values (not taking into account ga strains). when individual genomic regions within shseg were compared, differences among genotypes were observed: all hrsv a strains were identical at the ' utr of the g gene, but on strains showed a smaller percentage of conserved sites in the ' utr of the sh gene, compared to other regions. in na strains, the conservation percentage was similar throughout the shseg (differing by no more than . %, excluding the ' utr of the g gene). among ba strains, a smaller percentage of conserved sites was again observed in the ' utr of the sh gene, while the values for other regions were comparable. unlike group a, the ' utr of the group b g gene was not completely conserved, but the only difference among strains was in the last nucleotide of this region; % of strains had a cytosine and % an adenine. the strains analysed in this study possessed and different sh protein sequences in the hrsv a and hrsv b strains, respectively (fig. ) . the sh protein sequences identical to the group a or b consensuses was obtained in . % of strains. the others differed from the consensuses in only one amino acid (fig. ) , with two exceptions: a) the ga strains differed in amino acids; b) in a single strain, rsv - belonging to genotype na , amino acids - are missing. a single n-glycosylation site was predicted for all strains (the same one, fig. ); no potential o-glycosylation sites were recognised. in the intracellular and transmembrane protein region (amino acids - ), variability was seen only in group a strains and out of observed differences were such that differing amino acids had the same identity as corresponding amino acid in group b strains (shaded areas in fig. ). sequence coding for the fifth amino acid difference, s f, observed in only one on strain (hr - from ), presents so far a unique sh gene sequence, when compared to all sequences available in open public databases. greater versatility, including the deletion of amino acids, was observed in the sh ectodomain. again, two amino acid differences observed within hrsv a strains were such that differing amino acids had the same identity as the corresponding amino acids in group b strains (shaded areas in fig. ). among the hrsv b strains, differences in the amino acid sequence were seen only within the sh ectodomain. out of amino acid differences observes in the sh ectodomain (in both groups, fig. ), were in protein region - . the codon-based analysis of selective pressure did not identify any positively selected codon and the mean dn/ ds values were low: . , . and . for the na , on and ba genotypes, respectively, indicating that substitutions are not favoured and are purified from the population. the last analysis was restricted to strains identical in hvr nucleotide sequences and differing in sh protein (table ), in of them the differences were in the sh ectodomain. from those , in groups ( hrsv a and hrsv b), the differences were located within the sh protein region - , including the case where these amino acids were missing. due to the fact that sh gene utrs are relatively long (when compared to the coding region of the gene) and that the majority of mutations occur in them, hrsv sh gene is characterized by high variability while the sh protein remains highly conserved. besides the complete sh transcript, the genomic segment we analysed also included its two downstream regions, stopping immediately before the start codon of the g protein mrna. the last two regions were included because they are known to play a role in the transcriptional regulation of both upstream and downstream genes [ ] [ ] [ ] . we analysed whether a difference in the diversity of the sh/g junction (i.e. of the sequences between the stop codon of the upstream gene and the start codon of the downstream gene) among hrsv genotypes could be observed. a sequence characteristic only for ga strains was observed, but not all previously reported ga strains possess this sequence [ ] . whether it represents an evolutionary novelty, specific for recent ga strains requires further investigation (the oldest ga strain in our analysis was from mexico, from ). the overall nucleotide diversity of shseg was investigated using two methods: conservation percentage that is not influenced by the number of identical sequences; and p-distance that is, and therefore, defines more precisely diversity within this particular hrsv population. the results of both methods were concordant. the level of genetic diversity observed in shseg was not comparable to the one detected in hvr , except in the ' utr of the genotypes with prolonged g gene (on and ba ) which gained striking dominance within the short timespan we analysed. on the contrary, the other sh utr and sh-g intergenic region showed less variability in on and ba strains, when compared to na genotype. the current hrsv genotyping and molecular surveillance system is based on hvr [ , ] , a quite short genomic segment. it partitions hrsv strains in over group a and over group b genotypes. regarding phylogeny, shseg in not as informative as hvr . e.g. in analyses based on hvr , on and na strains are placed on different branches, even when one copy of the duplicated segment is excluded from the analysis [ ] . still, basing molecular epidemiology studies only on hvr may be quite misleading. in our pool of hrsv strains, over % of them would be incorrectly assumed to be identical to another strain if only the hvr region was analysed. an alternative hrsv genotyping scheme has recently been proposed [ ] , based on the complete g protein ectodomainwhich is twice longer than hvr . although this would increase the discrimination between strains, it is still focused solely on one protein gene. the g gene is non-representative of the hrsv genome, as it is marked by elevated substitution rates (attributed to relaxed selective constraints [ ] ) and a flip-flop substitution pattern [ ] . broadening the target genome sequence to sh (whose length in our analysis was - nts in group a, or in group b) or only to sh ' utr (in our analysis, this was a region of or nts in group a, in group b) would provide better insight into the interrelation between molecular epidemiology and molecular and evolutionary dynamics. after the translation of our genomic sequences, only different protein sequences were obtained. although group a and group b strains diverged approximately years ago [ ] , the majority of amino acids which differ from the consensus in group a have the same identity as corresponding amino acids in group b strains. this indicates that the variability of the sh protein is not only low, but is also restricted. based on the fact that hvr has the highest evolutionary rates, we concentrated on strains possessing the same hvr nucleotide sequences, but still differing in their sh protein sequences. those are the strains most likely to be highly similar and therefore we hypothesized that the observed differences could be a result of selective pressure. out of sets of strains with differences in the sh ectodomain, in sets the differences were in sh protein region - . in fact, besides s f the only amino acid differences observed in group a strains which differed from the group b consensus, were located within - region. in our viral pool, which was very limited both geographically and temporally, . % of group a strains and . % of group b strains possessed mutation in region - . our hypothesis that sh region - is an antigenic site still needs confirmation, but the same amino acids were shown to be highly variable by lima et al. [ ] . their analysis was performed on strains belonging to hrsv genotypes different from ours and detected in brazil in - . besides finding t i variability (as observed by us), p and a at position were also reported among group b strains. chen et al. [ ] detected variability in the same region in american hrsv strains from to [ ] . in group a, the most common amino acid at position was v (the same as in our analysis), but i was also observed. among group b strains, at position , besides the most common amino acid c and f (the same as in our analysis), chen et al. also identified y [ ] . a deletion of nts coding for amino acids - was found in the sh gene sequence of an na strain from january . the deletion was found only in this strain that caused a bronchiolitis in a -month-old child. the deletion was confirmed by independent rna isolations and preparations of dna for sequencing. during the same hrsv epidemic, strain rsv - (from november ) was detected in a sample from a -month-old child with bronchiolitis. the two strains are identical in the sh, g an f genes, except for the fact that rsv - does not possess this deletion in the sh gene. other genomic regions were not sequenced. in group a strains, h (which is missing from rsv - ) is a crucial residue in the regulation of sh ion channel activity [ ] . together with h , h plays a key role in the opening and closing mechanism of the sh pentameric pore, since they are located in strategic places within the chains, close to the n-and c-termini [ , ] . an alternative amino acid at position (y, a non-basic amino acid) has been reported before, in a brazilian ga strain from [ ] , but so far there were no reports of the deletion observed in rsv - . in vitro experiments regarding the functionality of the sh protein from rsv - or modelling studies that would compare its structure to other hrsv sh proteins have not been performed. basing hrsv molecular epidemiology studies solely on hvr largely underestimates the complexity of circulating virus populations. in strain identification, broadening of the genomic target sequence to sh gene would provide a more comprehensive insight into viral pool versatility. unlike its gene, sh protein is characterized by intra-group conservation and by highly restricted inter-group variability except for amino acids - , implicating that this protein region may be relevant for the antigenicity of the virus. all sequences are presented as plus strands, in the ′ to ′ orientation. additional file : figure s . phylogenetic tree of rsv strains based on hvr genomic segment. tree was generated using maximum-likelihood method, based on the general time reversible model and discrete gamma distributed rates across sites. the scale bar indicates the proportion of nucleotide substitutions per site. numbers are percentages of bootstrap values determined for iterations, only values above % are shown. strain designations are composed of ncbi genbank acc. no., name and genotype. (pdf kb) additional file : table s . sequences identical at hvr or shseg. (pdf kb) abbreviations dn/ds: the ratio of number of non-synonymous substitutions per nonsynonymous site and synonymous substitutions per synonymous site; f: fusion protein; g: glycoprotein; hrsv: human respiratory syncytial virus; hvr : the second hypervariable region of the glycoprotein gene; ncbi: national center for biotechnology information; nts: nucleotides; sh: small hydrophobic; slac: single-likelihood ancestor counting method; utr: untranslated region funding this work was supported by a grant from the croatian science foundation (project number to df), by university of zagreb (to ji-j) and by the grant "strengthening the capacity of cervirvac for research in virus immunology and vaccinology", kk. . . . . , awarded to the scientific centre of excellence for virus immunology and vaccines and co-financed by the european regional development fund. the datasets analysed during the current study are included in the published article and its additional file. taxonomy of the order 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differently to m -mediated antitermination variations in transcription termination signals of human respiratory syncytial virus clinical isolates affect gene expression variations in intergenic region sequences of human respiratory syncytial virus clinical isolates: analysis of effects on transcriptional regulation genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in argentina circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community nikic hecer a. early evolution of human respiratory syncytial virus on strains: analysis of the diversity in the c-terminal hypervariable region of glycoprotein gene within the first . years since their detection conservation of g-protein epitopes in respiratory syncytial virus (group a) despite broad genetic diversity: is antibody selection involved in virus evolution? genetic variability among complete human 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antivirals? not applicable. the authors declare that they have no competing interests. key: cord- - qt eth authors: cao, liyan; ge, xuying; gao, yu; herrler, georg; ren, yudong; ren, xiaofeng; li, guangxing title: porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: qt eth background: the lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (pedv) pathogenesis. vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv. nonetheless, the target cells of pedv in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. the immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. type i interferon (ifn) plays an important role in antiviral immune response. limited reports showed that pedv could inhibit type i interferon production. in this study, porcine small intestinal epithelial cells (iecs), the target cells of pedv, were used as the infection model in vitro to identify the possible molecular mechanisms of pedv-inhibition ifn-β production. results: pedv not only failed to induce ifn-β expression, but also inhibited dsrna-mediated ifn-β production in iecs. as the key ifn-β transcription factors, we found that dsrna-induced activation of ifn regulatory factor (irf- ) was inhibited after pedv infection, but not nuclear factor-kappab (nf-κb). to identify the mechanism of pedv intervention with dsrna-mediated ifn-β expression more accurately, the role of individual molecules of rig-i signaling pathway were investigated. in the upstream of irf- , tank-binding kinase (tbk )-or inhibitor of κb kinase-ε (ikkε)-mediated ifn-β production was not blocked by pedv, while rig-i-and its adapter molecule ifn-β promoter stimulator (ips- )-mediated ifn-β production were completely inhibited after pedv infection. conclusion: taken together, our data demonstrated for the first time that pedv infection of its target cell line, iecs, inhibited dsrna-mediated ifn-β production by blocking the activation of ips- in rig-i-mediated pathway. our studies offered new visions in understanding of the interaction between pedv and host innate immune system. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, rna virus of coronaviridae family, which is the main etiological agent of severe diarrhea in pigs of all ages and fatality in neonates [ ] . outbreaks of porcine epidemic diarrhea (ped) have received extensive attention for the considerable economic losses to the swine industry worldwide. great advances have been made in elucidation of the molecular epidemiology, diagnosis, prevention, and treatment of ped [ ] . recently, coronavirus interaction with host innate immune system has been a hot research field. previous studies indicated that transmissible gastroenteritis virus (tgev) infection enhanced type i interferon expression and its protein modulated type i ifn expression [ , ] . for mouse hepatitis virus (mhv), ifn production among different cell populations varied due to their diverse susceptibility to this virus [ ] [ ] [ ] [ ] [ ] . furthermore, both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) do not induce type i ifn (ifn-α/ β) activation [ ] [ ] [ ] . so far, limited reports showed that pedv could inhibit type i interferon production [ , ] . during viral infection and replication, the host innate immune response is the first line of defense; therefore, the ability of viruses to suppress or avoid this response is crucial for their pathogenic potential. ifn-α/β is an essential element of the host innate immune response against viral infections. double-stranded rna (dsrna), the replicative intermediate of most viruses, is a potent inducer of ifn-β, which is recognized as a pathogen-associated molecular pattern (pamp) by host pattern recognition receptors (prrs). two of major prrs, retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene (mda ) detect dsrna in the cytoplasm [ ] . following dsrna binding, rig-i and mda recruit corresponding adapter protein ifn-β promoter stimulator (ips- ) that, in turn, activate downstream signaling of tank-binding kinase (tbk ) and inhibitor of κb kinase-ε (ikkε) transduction, leading to the activation of transcription factor ifn regulatory factor (irf- ) and nuclear factor-kappab (nf-κb). activated irf- , and nf-κb bind to ifn-β enhancer and initiate ifn-β transcription [ ] . vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv [ ] . however, it was often considered that vero cells might lack genetic component necessary for ifn production [ ] [ ] [ ] . porcine intestinal epithelial cells (iecs) are thought to the target cells of pedv, which play an important role in the activation of host immune responses by induction of key signaling molecules, including cytokines, surface molecules, and chemokines during microoganism invasion [ , ] . in the present study, to determine if pedv infection suppresses ifn-β activation, we chose iecs as an infection model to research the molecular mechanisms of pedv infection and the host antiviral innate immune response. our results clearly suggested that pedv prevented dsrna-induced ifn-β synthesis by blocking rig-i-mediated pathways. pedv failed to induce ifn-β expression and inhibited poly (i:c)-mediated ifn-β production in iecs type i ifns (ifn-α/β) are critical to the host antiviral innate immune response. however, there is no evidence suggesting that iecs produce type i ifns in response to pedv infection. previous studies have showed that pedv could be propagated in iecs [ , ,] . to confirm whether pedv infection could induce ifn-β production in iecs or not, we transiently cotransfected the ifn-β/ luciferase reporter plasmid (ifn-β-luc) and the renilla luciferase construct phrl-tk and then infected with pedv (at an moi of or . , respectively) or mockinfected for h. the cells were retransfected with μg of poly (i:c) as a positive inducer. as shown in fig. a , ifn-β luciferase activity enhanced markedly in positive controls, while it was almost not detected in pedv-infected iecs. in addition, ifn-β mrna expression was hardly detected in pedv-infected iecs similar to mock-infected group, however, it had significant expression in poly (i:c)-transfected group at the indicated times ( h and h, p < . ) (fig. b) . this result was consistent with the luciferase reporter assay. taken together, pedv infection of iecs did not induce ifn-β activation. increasing evidence showed that viruses not only inhibit the induction of type i ifns, but also block dsrnainduced production of type i ifns to escape the innate immune surveillance of the host [ ] [ ] [ ] . to identify whether pedv was able to inhibit dsrna-induced ifn-β production, ifn-β-luc was transfected into pedv-infected and uninfected cells, respectively. the cells were retransfected with or without poly (i:c) h later. as a result, activation of the ifn-β promoter decreased significantly in poly (i:c)-transfected, pedvinfected cells compared with mock-infected cells transfected with poly (i:c) (fig. c) . it showed pedv also inhibited poly (i:c)-mediated ifn-β induction. pedv impeded poly (i:c)-mediated activation of irf- , but not nf-κb irf- and nf-κb are two essential ifn-β transcription factors. in unstimulated cells, irf- is ubiquitously present in the cytoplasm as an inactive monomer, whereas nf-κb is present as a homodimer or heterodimer bound to the inhibitory proteins iκb in the cytoplasm [ ] . phosphorylation, which is a key step during irf- and nf-κb activation, in turn, leads to nuclear translocation. therefore, we evaluated whether pedv infection induced irf- and p activation by western blot analysis. following pedv infection, the whole cell extracts were prepared for the indicative times, in fig. a , irf- still existed in the cytoplasm, and the levels of irf- protein was almost equal with mock-infected cells, while the phosphorylation of irf- (p-irf- ) did not detect in the pedv infected cells in comparision to a obviously signal in poly (i:c)-transfected cells. on the contrary, compared with the amount of nf-κb subunit p in the cytoplasm, the phosphorylation of p (p-p ) increased with progression of pedv infection in the nucleus. meanwhile, the concentration of poly (i:c)induced p nuclear translocation was clearly increased. pedv n protein was also detected in pedv-infected cells. these data suggested that pedv did not induce activation of irf- , but nf-κb. we then used luciferase reporter assay system to determine whether irf- and nf-κb are linked with the inhibition of ifn-β production after pedv infection. as shown in fig. b , irf- luciferase activity was sharply decreased in pedv-infected cells, and poly (i:c)-induced irf- activation was also inhibited by pedv in comparison to a remarkably signal in poly (i:c)-transfected cells. however, in fig. c , compared with mock-infected to detect the activation of irf- and p after pedv infection, the cell extracts were prepared at the indicated times and subjected to western blot analysis with antibodies specific for irf- , p , p-irf- , p-p and pedv n mcab. anti-β-actin was included as a control for sample loading. these experiments were performed in duplicate. b and c iecs were infected or mock-infected with pedv at an moi of for h, and then cells were cotransfected with (prdiii-i) -luc (b) or pnf-κb-luc (c) and phrl-tk for additional h. cells were retransfected with poly (i:c) for h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. **p < . as compared with poly (i:c). d irf -gfp fusion protein transfected with iecs and then infected with pedv at an moi of and mock-infected cells served as negative controls. h later, cells were retransfected with poly (i:c) (positive control) (c and d) or untransfected (a and b) for h. cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and stained by dapi (blue). cells were incubated with anti-pedv rabbit polyclonal antibody (red) and tritc-labeled goat anti-rabbit secondary antibody, then analyzed for fluorescence by confocal microscopy. magnification, × (leica, wetzlar, germany) (see figure on previous page.) fig. pedv does not induce ifn-β production and inhibits poly (i:c)-mediated ifn-β induction. a iecs were cotransfected with ifn-β-luc and phrl-tk, then infected with pedv at an moi of and . for h. cells were retransfected with poly (i:c) as a positive control. after h, the cells were harvested and subjected to a dual-luciferase assay. b iecs were infected with pedv at an moi of , mock-infected as a negative control, or transfected with poly (i:c) as a positive control. at the indicated time points, total rna was extracted and ifn-β and β-actin mrna were subjected to real-time pcr. rna expression levels were normalized to β-actin. c in contrast to a, iecs were first mock-infected or infected with pedv at an moi of for h and then cotransfected with ifn-β-luc and phrl-tk for h. cells were retransfected with or without poly (i:c) for an addition h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. *p < . ; **p < . as compared with poly (i:c) cells, nf-κb luciferase activity significantly enhanced both in pedv-infected and poly (i:c)-transfected cells. in addition, we found that poly (i:c)-induced activation of nf-κb was not blocked by pedv. to further identify pedv-inhibited poly (i:c)-mediated activation of irf- , confocal microscopy assay was used. as a result, irf -gfp remained in the cytoplasm of both mockinfected ( fig. d. a) and pedv-infected (fig. d . b) iecs compared with poly (i:c) controls, in which clear translocation to the nucleus was observed (fig. d. d) . furthermore, pedv could block poly (i:c)-mediated irf- nucleus migration (fig. d. c) . taken together, our date clearly implied that pedv impeded dsrna-mediated ifn-β transcription primary by interfering with irf- activation, but not nf-κb. pedv failed to block tbk /ikkε activity tbk and ikkε are essential kinases for the irf- activation [ ] . in order to ascertain whether pedv inhibited poly (i:c)-induced irf- activation by impeding tbk /ikkε kinase activity, we cotransfected plasmids expressing tbk /ikkε kinase and a plasmid encoding the ifn-β promoter of the luciferase reporter into infected or mock-infected iecs and retransfected the cells with or without poly (i:c) at h.p.i. as show in fig. , tbk /ikkε overexpression increased ifn-β promoter activity in both infected and mock-infected iecs, and obviously upregulation was detected in iecs transfected with poly (i:c), suggesting that pedv failed to block tbk /ikkε activity. however, poly (i:c)-induced ifn-β promoter activity in iecs overexpression of tbk / ikkε plasmids was significantly inhibited by pedv. it showed that pedv interrupting dsrna-induced ifn-β production should localize upstream from tbk /ikkε. it is possible that pedv blocks poly (i:c)-mediated ifnβ production by suppression of the individual molecules upstream of tbk /ikkε in rig-i signaling pathway. to explore this possibility, mock-and pedv-infected iecs were cotransfected with ips- expression plasmid and ifn-β promoter luciferase reporter plasmid, respectively. as shown in fig. a , overexpression of ips- in iecs could enhance ifn-β luciferase activity in mock-infected cells, but it was completely inhibited in pedv-infected cells. for the poly (i:c) transfection experiments, there appeared significant restriction of ifn-β luciferase expression in pedv-infected cells compared with that of mock-infected cells. these date indicated that pedv interacted with ips- to block poly (i:c)-mediated ifn-β transcription. ips- is an adapter molecule of rig-i, the data showed that pedv blocked ips- -induced ifn-β production in dsrna signaling pathway, thus, we speculated that rig-i-induced ifn-β production in this signaling pathway was also inhibited. to verify it, mock-and pedvinfected iecs were transfected with rig-i expression and ifn-β reporter plasmids. the results showed that ifn-β luciferase activity was markedly increased in iecs overexpressing rig-i, but was completely inhibited by pedv infection. and the ifn-β reporter signal could be observed in iecs stimulated with poly (i:c), while this signal could be sharply reduced in rig-itransfected, poly (i:c)-stimulated and pedv-infected iecs (fig. b) . in summary, the findings of the present study suggested that pedv-infection in iecs inhibits dsrna-induced ifn-β induction by interfering with irf- activity associated with rig-i-mediated signaling pathway. the target interaction molecules of pedv intervention of dsrnainduced ifn-β production primarily was ips- . however, as a limitation to this study, host cells may inhibit or activate multiple signaling pathways simultaneously in response to exogenous stimulus, and some other transcription factors may have been blocked or activated in this process. here, we only addressed the mechanisms of pedv-induced inhibition of ifn-β production in relation to the molecules of rig-i signaling pathways in vitro. further studies are needed. overall, elucidation of the cells were harvested and subjected to a dual-luciferase assay. data were analyzed and the ratio of firefly luciferase expression to renilla luciferase activity was shown. all data are expressed as means ± sd of independent experiments. **p < . compared with pedv-infected, expression plasmids or vector-transfected control influence of pedv evasion of the host innate immune response will aid in the development of antiviral agents to prevent the spread of pedv during the early infection phase. the african green monkey kidney cell line veroe and swine small intestine epithelial cells (iecs) [ , ] were respectively cultured in dulbecco's modified eagle's medium (dmem) and dulbecco's modified eagle's f ham medium (dmem-f ) supplemented with % fetal bovine serum at °c in a humidified atmosphere of % co . pedv strain cv was propagated in veroe cells in dmem containing . μg/ml of trypsin. poly (i:c) was purchased as a sodium salt (sigma-alorch, saint louis, mo, usa) and dissolved in water to obtain a stock solution of mg/ml. the dual-luciferase® reporter assay system was purchased from promega corporation (madison, wi, usa) and monoclonal anti-β-actin antibody was purchased from sigma-aldrich (st. louis, mo, usa). anti-irf- , anti-p anti-p-irf- and anti-p-p rabbit polyclonal antibodies and secondary horseradish peroxidase (hrp)-conjugated anti-rabbit igg were purchased from cell signaling technology, inc. (beverly, ma, usa). rhodamine isothiocyanate (tritc)-labeled goat anti-rabbit igg were purchased from the zhongshan company (beijing, china). anti-pedv rabbit polyclonal antibodies and anti-pedv n protein monoclonal antibody (mcab) were prepared in our laboratory, which could specifically react with pedv. the plasmids ifn-β-luc for ifn-β, prd (iii-i) -luc for irf- , and pnf-κb-luc for nf-κb were kindly donated by dr. shaobo xiao (huazhong agricultural university, wuhan, hubei province, china) [ ] . the pef-bos empty vector and pef-flag-rig-i recombinant expression plasmid were kindly provided by t. fujita (tokyo metropolitan institute of medical science, tokyo, japan) [ ] . the pef-bos-flag-trif, pcdna -flag-ikkε and pcdna -flag-tbk recombinant expression plasmids, and the pcdna empty vector, and the conjugate irf -green fluorescence protein (gfp) expression construct were kindly provided by k. fitzgerald (university of massachusetts medical school, worcester, ma, usa) [ ] . the porcine ips- (ncbi accession no: eu . ) gene was cloned from porcine kidney cells by reverse transcription polymerase chain reaction (rt-pcr) using the specific primer pair. the ips- primers were ′-ccgggtaccaccatgacgtttgccgaggacaa- ′ and ′-tttctcgagtcactggggcaggcgccgcc- ′. porcine ips- was inserted into pcdna . (+) using the restriction enzymes kpni and xhoi. cells were harvested and luciferase activity was analyzed using a dual-luciferase assay. all data are expressed as means ± sd of independent experiments. **p < . compared with pedv-infected, expression plasmids or vector-transfected control when the cells reached %- % confluence. the cells were then infected or mock-infected with pedv for h. cells were retransfected with or without poly (i:c) ( . μg) for an additional h. or iecs were infected or mock-infected with pedv for h prior to transfection the luciferase reporter plasmids alone or cotransfection the indicated expression plasmids ( . μg). the cell lysates were harvested and luciferase activity was analyzed using a dual-luciferase assay system and a luminometer (turner biosystems, inc. sunnyvale, ca, usa) according to the manufacturer's instructions. data represent relative firefly luciferase activity normalized to renilla luciferase activity. the resulting ratios were used to compare the expression of the firefly luciferase gene in pedvinfected cells to that present in mock-infected cells. total rna was extracted from the transfected cells using triquick reagent (beijing solarbio science & technology co., ltd., beijing, china) according to the manufacturer's instructions and then reverse-transcribed into complementary dna (cdna) using murine leukemia virus reverse transcriptase (gbi labs/golden bridge international, inc., mukilteo, wa, usa) with oligo dt random hexamers (haigene technology, harbin, china). the cdna was then subjected to real-time pcr with specific primer pairs targeting ifn-β (f: ′-gctaac aagtgcatcctccaaa- ′ and r: ′-ccaggagc ttctgacatgcca- ′) and β-actin (f: ′-ggctcag agcaagagaggtatcc- ′, and r: ′-ggtctcaaa catgatctgagtcatct- ′. β-actin mrna was used as an endogenous control. iecs were seeded in -well plates and then transfected with μg of irf -gfp fusion expression constructs per well using lipofectin transfection reagent (invitrogen corp.) when cells reached confluence. cells were then mock-infected or infected with pedv at a multiplicity of infection (moi) of . at h postinfection, cells were transfected with μg of poly (i:c) or left untransfected. after h, cells were removed from the culture medium and washed three times in cold phosphatebuffered saline (pbs). next, cells were fixed in % paraformaldehyde for min at room temperature, quenched with . m glycine for min, and then permeabilized with . % triton x- for min. afterward, the cells were incubated with anti-pedv antibody (dilution, : ) for h followed by tritclabeled goat anti-rabbit secondary antibody (dilution, : ) for min at °c. the nuclei were stained with ′, -diamidino- -phenylindole-dihydrochloride (dapi) (invitrogen corp.). cells were examined using a tcs sp aobs confocal microscope (leica camera ag, wetzlar, germany). iecs were infected with pedv at an moi of or treated with poly (i:c) for the indicative times, lysed in × sodium dodecyl sulfate (sds) sample buffer and boiled for min. whole cells extracts were separated by % sds-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was blocked with % (w/v) bovine serum albumin (bsa) in tris-buffered saline ( mm tris-cl at ph . and mm nacl) containing . % tween (tbst) at room temperature for h. the membranes were then incubated with a primary antibody (dilution, : ) at °c overnight and a secondary hrp-conjugated antibody (dilution, : ) for h at room temperature. protein blots were developed using an enhanced chemiluminescence (ecl) detection system and exposed to x-ray film (clinx science instruments co., ltd., shanghai, china). all data were expressed as means ± standard deviations (sd) of independent experiments. the statistical significance was tested by student 's t-test and p-values less than . were considered statistically significant. porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs 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cells have a genetic defect in interferon production sars coronavirus and innate immunity first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine porcine epidemic diarrhea virus infection induces nf-kappab activation through the tlr , tlr , and tlr pathways in porcine intestinal epithelial cells porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway hepatitis a virus suppresses rigi-mediated irf- activation to block induction of beta interferon human rhinovirus attenuates the type i interferon response by disrupting activation of interferon regulatory factor induction of irf- /- kinase and nf-kappab in response to double-stranded rna and virus infection: common and unique pathways ikkepsilon and tbk are essential components of the irf signaling pathway porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and upregulates interleukin- expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank shaobo xiao, takashi fujita, kate fitzgerald for kindly providing important constructs. this work was supported by the programme for new century excellent talents at the heilongjiang provincial university all authors declare that there are no financial or other relationships that might lead to a conflict of interests.authors' contributions lyc response for carrying out the experiments, date analysis and drafting the manuscript. xyg and yg construct the recombinant expression plasmids. ydr participated in date analysis. gh, xfr and gxl designed the experiments and reviewed manuscript. all authors have seen and approved the manuscript and have contributed significantly to the work. key: cord- -kqftn t authors: sun, jia-zeng; wang, jigui; wang, shuang; yuan, daoli; li, zhili; yi, bao; hou, qiang; mao, yaping; liu, weiquan title: microrna mir- a and mir- inhibit mink enteritis virus infection by repression of its receptor, feline transferrin receptor date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: kqftn t mink enteritis virus (mev) is one of the most important pathogens in the mink industry. recent studies have shed light into the role of micrornas (mirnas), small noncoding rnas of length ranging from – nucleotides (nt), as critical modulators in the host-pathogen interaction networks. we previously showed that mirna mir- b can inhibit mev replication by repression of viral non-structural protein expression. here, we report that two other mirnas (mir- a and mir- ) inhibit mev entry into feline kidney (f ) cells by downregulating its receptor, transferrin receptor (tfr), by targeting the ′ untranslated region (utr) of tfr mrna, while being themselves upregulated. mink enteritis virus (mev) is an autonomous parvovirus that causes important disease in mink, leading to huge economic losses worldwide. mev is a single-stranded negative sense dna virus belonging to the family parvoviridae, with a genome of about kb containing main open reading frames (orfs). mev, a variant of feline panleukopenia virus (fpv) and highly homologous with canine parvovirus (cpv), causes a highly infectious acute disease of mink characterized by extensive virus replication in mesenteric lymph nodes and intestinal crypt epithelial cells, with an associated loss of intestinal mucosa, diarrhea, and a high rate of morbidity and mortality [ ] [ ] [ ] [ ] [ ] . micrornas (mirnas) are endogenous and highly conserved small noncoding rnas of length - nucleotides (nt), which have gained widespread attention as critical modulators in many biological processes including cell proliferation and differentiation, development and apoptosis. in animals, mirnas are imprecisely complimentary to their mrna targets and act by repression of target gene expression [ ] [ ] [ ] [ ] . attention has also been paid to the role of mirnas as effectors in host-virus interaction networks [ , ] , either by targeting cellular factors useful for virus replication [ , ] or by directly targeting virus mrnas [ ] [ ] [ ] [ ] . we have recently reported that a cellular mirna mir- b inhibits mev infection by repression of viral non-structural protein expression [ ] , which indicates that cellular mirnas may play a direct role on viral mrnas themselves. for many animal viruses, cell entry and infection are initiated by receptor-mediated endocytosis involving specific cellular surface components. transferrin receptor (tfr), is required for the import of iron into the cell, and is regulated by intracellular iron concentration. it has also been reported to be a receptor for mev, controlling the first step in the viral infection process. tfr can be considered a model for the endocytosis and recycling of receptor-ligand complexes: it is excluded from lipid raft domains in the plasma membrane and is taken up rapidly from the cell surface via clathrin-mediated endocytosis [ , [ ] [ ] [ ] [ ] [ ] [ ] . since tfr plays an important role in mev infection, we therefore investigated whether mirnas participate in the hostvirus interaction by modulating its activity. feline f cells, obtained from the american type culture collection (atcc), were cultured as monolayers in minimum essential medium (mem) (gibco, ca) containing % fbs (hyclone, logan, ut), and % penicillinstreptomycin (gibco) at °c in a % co atmosphere. mev strain l was originally isolated from an infected animal in a mink farm, liaoning province, china. the whole viral genome has been sequenced in our laboratory and found to have high identity with mev strain abashiri (genbank accession, d . ). for infection, f cell monolayers were first dispersed by . % trypsin and virus was added to the suspension before incubation in the original plates. deep sequencing of small rnas and analysis of the sequencing data [ ] f cells cultured in -well plates (costar) were infected with mev at an input multiplicity (moi) of pfu/cell. uninfected cells were maintained as a control. twenty-four h later, the triplicate cultures were pooled, total rna was extracted by trizol reagent (invitrogen) and small rnas with length of - nt were separated by page. ten μg samples of the isolated rnas were submitted to solexa (illumina) for sequencing as cdna libraries. duplicate sequences were eliminated from the initial data set. the resulting sets of unique reads were mapped onto the feline genome [ , ] using the program short oligonucleotide analysis package (soap) [ ] . perfectly matched reads were also mapped onto the mirnas of six reference species (homo sapiens, canis familiaris, mus musculus, rattus norvegicus, bos taurus and sus scrofa) listed in the sanger mirbase (release ) using the patscan tool [ ] to identify homologs of known mirnas. rnahybrid tools (http://bibiserv.techfak.uni-bielefeld.de/ rnahybrid/submission.html/) [ ] were used to predict mirna targets in tfr mrna ′utr following the rules of no mismatch and g/u complementarity in mirna seed sequences. regrna tools (http://vita.mbc.nctu.edu.tw/) [ ] were also used to predict regulatory rna motifs in the tfr mrna ′utr. targetscan (http://www.targetscan. org/) tools were used to predict conservative mirna targets in the tfr mrna ′utrs of different species. the luciferase expression vector pgl -control (promega) was used for construction of predicted mirna candidate targets containing a luciferase reporter gene, with prl-tk (promega) as control. sequences containing part of the tfr ′utr and candidate targets of mirnas were amplified by rt-pcr and directionally inserted into the ′utr of the luciferase gene in the pgl -control vector, generating pgl -tfr ′utr. to facilitate cloning, the first pcr product amplified by the first pair of primers ( ′-atgtggtacctatacttatatgagaac- ′ and ′-tccgtgttcaagcattttattaaatc- ′) was used as a template and an xba i restriction site (italics) was added to the '-( ′-gctctagaatgtggtacctat acttatatgagaacagc- ′) and ′-( ′-gctctaga tccgtgttcaagcattttattaaatcag- ′) secondary pair of primers. to further ascertain that the binding sites of the predicted mirnas in tfr ′utr indeed existed, tetranucleotide mutations were generated in the two potential target sites of pgl -tfr ′utr using pcr, resulting in mut a-pgl -tfr ′utr ( ′-cactag atttctttaggcagcacgaattaatacagggtagg tac- ′ and ′-gtacctaccctgtattaattcgtgc tgcctaaagaaatctagtg- ′) and mut -pgl -tfr ′utr ( ′-cttcaagttaaagtgaataaggtgt taaaaatgttcatgatagaatc- ′ and ′-gattct atcatgaacatttttaacaccttattcactttaact tgaag- ′). mutant plasmids were generated by pcr using primestar max dna polymerase (takara), ng of the parent vectors as templates and the complementary primers under the following conditions: °c for min, followed by cycles of °c for s, °c for s and °c for s, followed by °c min. the resulting products were digested with μl dpn- for h at °c to remove the parental dna. the remaining dna was used to transform competent dh α cells, and a number of colonies containing mutant plasmids were obtained and confirmed by sequencing (shanghai sangong co.). the mir- a, mir- b, mir- , mir- , mir- , mir- and mir- mimics and inhibitors, mut mir- mimics (in which the tetranucleotide mutation was complementary to mut pgl -tfr ′utr) and mut mir- mimics (in which the tetranucleotide mutation was complementary to mut pgl -tfr ′utr) were synthesized by genepharma, shanghai. all mimics were double-stranded rna oligos, while inhibitors were singlestranded. negative control mimics and inhibitors were also synthesized for control experiments. to determine whether the selected mirnas play a direct role in repression of luciferase expression from pgl -tfr ′utr, -well plates seeded with f cells at - % confluence were co-transfected with a mixture of pgl -tfr ′utr ( μg/ml) and prl-tk vector ( μg/ml) together with mimics ( nm). the mut a or mut pgl -tfr ′utr and prl-tk together with mimics ( nm) were co-transfected to verify accuracy of the seed sequence. nc mimics were used as negative controls. after h, the cells were harvested for relative luciferase activity assay. to determine the effects of the selected mirnas on mev infection, f cells, at a confluence of - % in -well plates, were transfected with mimics ( nm). after h, the cells were dispersed with . % trypsin and infected with mev (moi = . ). virus infection was measured by qpcr and flow cytometric analysis at the indicated times. to quantify the relative luciferase activity, a dual-luciferase reporter assay system kit (promega) was used according to the manufacturer's protocol. co-transfected cells with a mixture of luciferase reporter plasmids were washed with cold phosphate-buffered saline (pbs). passive lysis buffer (promega: μl) was then added to the cells in each well. after min, the supernatants were clarified by centrifugation at , g for s, and the luciferase activity was measured using a modulus single-tube multimode reader (promega). relative luciferase expression was calculated as the expression of firefly luciferase (pgl -control vector) divided by that of renilla luciferase (prl-tk). to detect whether selected mirnas can downregulate the expression of tfr, qpcr analysis was performed. after transfection of f cells with mirna mimics or inhibitors, nc mimics or inhibitors as controls, total rna was extracted and digested with dnase i (takara). two μg total rna of each sample was reverse transcribed using m-mlv reverse transcriptase (promega) according to the manufacturer's protocol. the β-actin mrna level was measured as a control. primers used for amplification were: β-actin, ′-cgggacctgacggactacct- ′ and ′-ggccatctcctgctcaaaat- ′ and tfr, ′-atg attggctacttgggctattg- ′ and ′-cctgatgg tgctggtgaactc- ′. to detect the viral genomic dna quantitative level in f cells, total dna was extracted and the concentration was measured. pcr amplication of a fragment of viral genomic dna ( ′-gcttacgctgcttatcttcgc- ′, ′-taatgtcctattttccccccc- ′) was performed. to determine the expression level of mirnas in f cells, total rna was extracted, and μg was polyadenylated using e. coli poly (a) polymerase according to the manufacturer's protocol (promega). the poly (a) reaction product was then reverse transcribed using m-mlv reverse transcriptase (promega) and an adaptor primer [ ] ( ′-gcgagcacagaattaatacgactcactat aggttttttttttttvn- ′) according to the manufacturer's protocol. pcr amplication was carried out using the specific mirnas primers (mir- a, ′-aaaagcg gggagagggcg- ′ and ′-gcgagcacagaattaat acgactcac- ′ and mir- , ′-cagtggttttacc ctatggtagaaa- ′ and ′-gcgagcacagaattaa tacgactcac- ′). u small rna expression level was measured as a control using primers ′-ctcgctt cggcagcaca- ′ and ′-aacgcttcacgaatttg cgt- ′. cycling conditions for qpcr using fastsybr mixture (cwbio) and the viia™ real-time pcr system (applied biosystems) were °c for s, followed by cycles of °c for s and °c for s. the data were analyzed by the ΔΔct method [ ] . western blot assay f cells transfected with mimics in a -well plate were washed times with cold pbs, a mixture of μl ripa lysis buffer (hx-bio) and . mm psmf was added and the cells were harvested into eppendorf tubes. after min on ice and centrifugation at , g for min, μl supernatant was mixed with μl each × sds sample buffer and boiled for min. samples were subjected to % sds-page gel and transferred to a nitrocellulose membrane (pall life science). the membranes were blocked with % nonfat dry milk for h, then incubated for h at room temperature with purified primary mouse antibody cd (h . ) (santa cruz: : dilution) or anti-β-actin antibody (mbl: : , dilution) in nonfat milk. after washes with tris-buffered saline containing . % tween- (tbst), the membranes were incubated for h at ambient temperature with the appropriate horseradish peroxidase-conjugated secondary antibody (mbl: : , dilution) in tbst. protein bands were visualized using ecl western blot substrate (thermo), with βactin as a control. treated f cell monolayers were dispersed with . % trypsin, harvested and fixed in % paraformaldehyde. after washes with pbs and incubation for h at °c with anti-cd mouse antibody ( : ) or anti-mev rabbit polyclonal antibody (prepared in this laboratory) at : , the cells were washed times with pbs, incubated with fluorescein isothiocyanate (fitc)-conjugated goat antimouse or anti-rabbit igg antibody (mbl: : dilution) for h at °c, washed another times with pbs and analysed by bd facscalibur flow cytometry. nonspecific rabbit polyclonal antibody (iso) (prepared in this laboratory) was used as an isotype control. the data were analyzed using bd cellquest software. human anti-ago antibody (abnova) was first bound to protein a/g-agarose (abmart) in pbs for min at °c. treated f cells were harvested, washed and solubilized in ripa lysis buffer (hx-bio) and psmf for min on ice, then centrifuged at , g for min to clarify the supernatant. the latter was then added to the ago /agarose conjugate and incubated for h at °c. incubation of the supernatant with normal mouse igg (mbl) was used as a negative control. rna bound to the ago protein was dissociated with trizol reagent and reverse transcribed. tfr, mir- a and mir- were quantified by qpcr analysis, with β-actin and u small rna as internal controls. data were analysed statistically using graphpad software, as described in the figure legends. screening of mirnas targeting tfr mrna ′utr as described in materials and methods, small rna ultrahigh throughput sequencing was performed (solexa) on uninfected f cells and following mev infection (moi = ) to detect mirnas targeting tfr ′utr. two mirna libraries were also constructed [ ] . screening for mirnas with rnahybrid [ ] , regrna [ ] and targetscan tools identified mirna candidates (figure ). to test these mirnas, f cells were transfected with the mirna mimics and inhibitors, negative control (nc) mimics and inhibitors as controls. after h, tfr mrnas were quantified by qpcr. results showed that mir- a and mir mimics decreased tfr mrna levels by almost % compared to nc mimics, and mir- a inhibitors increased them approximately % (figure a ). after h transfection as above, the quantity of tfr proteins were also examined by western blot and flow cytometry. results showed that mir- a and mir- downregulated the protein level ( figure b ) and the percentage of tfr-positive cells by almost % and % respectively ( figure c ). to further confirm the activity of the mimics, qpcr and western blot assay were used to show that the expression levels of tfr were reduced in a dose-dependent manner ( figure d,e) . altogether, these results clearly show that cellular mir- a and mir- have negative effects on tfr expression. the expression levels of predicted mirnas are shown in table . following mev infection both mir- a and mir- showed relatively higher level expression and greater upregulation than found in uninfected cells. this was confirmed by qpcr which showed that both mirnas gradually increased following mev infection ( figure ). to investigate whether upregulation of the two mirnas following mev infection could affect tfr expression, qpcr ( figure a ,b) and western blot ( figure c ) were performed simultaneously. as expected, tfr was gradually downregulated following mev infection. to further ascertain that tfr downregulation is the result of mirnas upregulation, f cells were transfected with mirna inhibitors h post mev infection. results showed that compared with transfection with nc inhibitors, both mir- a and mir- inhibitors attenuated the inhibitory effect on tfr expression levels ( figure d ). it appears, therefore, that upregulation of mir- a and mir- directly results in tfr downregulation. mir- a and mir- target the ′utr of tfr and physically bind to tfr mrna in the risc to confirm that mir- a and mir- directly target tfr ′utr and show inhibitory activity, a reporter vector pgl -tfr ′utr containing the potential target (d) qpcr was used to assess the tfr mrna levels after h transfection with mir- a and mir- mimics in a dose-dependent manner (e) western blot assay was used to assess the tfr protein levels after h transfection with mir- and mir- mimics in a dose-dependent manner. data are from independent experiments (mean ± sd). statistical significance was analyzed by student's t test; *p < . ; **p < . ; ns, not significant. segment in the ′utr of the luciferase gene was constructed ( figure a ). after co-transfection of f cells with pgl -tfr ′utr and mir- a or mir- mimics, the relative luciferase activity was measured. as predicted, both mir- a and mir- significantly inhibited the relative luciferase activity (by more than half ) in comparison with nc mimics ( figure b ). to further ascertain the necessary function of complementary seed sequence, mut a-pgl -tfr ′utr and mut -pgl -tfr ′utr, with mutated tetranucleotides were constructed and the corresponding mut mir- a and mut mir- mimics were synthesized ( figure a ). as expected, after co-transfection of f cells, mir- a and mir- mimics had no effect on the relative expression activity of the mutant reporter vector, but mut mir- a and mut mir- mimics downregulated them by more than -fold ( figure c,d) . to provide evidence for the physical interaction of the two mirnas with tfr mrna, immunoprecipitation with argonaute (ago ) was performed. as previously described [ ] , both mrna degradation and translation repression is dependent on risc, in which the most important factor is ago protein. therefore, anti-ago antibody was used to test if the two mirnas and tfr mrna were associated with ago . as expected, when ago protein was specifically precipitated by anti-ago antibody, as compared with normal igg, tfr was enriched more than -fold by mir- a and more than -fold by mir- after transfection with mir- a or mir- mimics respectively, whereas following transfection with mut mir- a or mut mir- , tfr was not enriched ( figure e ). altogether, these results indicate that mir- a and mir- target the ′utr of tfr and physically bind to tfr mrna in the risc. since mir- a and mir- downregulated tfr expression, we speculated that the two mirnas could control mev infection by preventing the virus from entering cells. to investigate this, f cells were transfected with either mir- a and mir- mimics, with nc mimics as controls. after h transfection, the cells were infected with mev (moi = . ). at the indicated times, the quantity of viral genomic dna was measured. as predicted, results showed that mir- a and mir- downregulated the quantity of viral genomic dna in f cells, even during the early stage of virus infection ( figure a ). to further validate the results above, flow cytometry was used to determine the proportion of mev-infected cells. results showed that compared with transfection with nc mimics, both mirnas mimics had a negative effect on mev-infected cell numbers from quite early on ( figure b,c,d,e) . these results clearly demonstrated that mir- a and mir- inhibit mev infection by preventing virus entry into the cells. mir- a and mir- play roles on tfr and mev in a synergistic manner as described above, both mir- a and mir- inhibit mev infection by downregulating tfr expression. since the targets in tfr ′utr of the two mirnas are different, we speculated that the two mirnas could show function together. to investigate this, f cells were transfected with mir- a and mir- mimics individually or together, with nc mimics as controls. after h transfection, tfr mrnas were assayed by qpcr. results showed that mir- a and mir mimics together decreased tfr mrna levels by almost % compared to mir- a or mir- mimics ( figure a ). at h, western blot assay also showed that the decrease in tfr protein level was enhanced by the two mirnas together ( figure b ). to confirm that the two mirnas also showed greater negative function on mev infection, f cells were transfected with mir- a and mir- mimics individually or together, with nc mimics as controls. after h transfection, the cells were infected with mev (moi = . ) and (see figure on previous page.) figure mir- a and mir- inhibit mev infection by preventing the virus to entry into f cells. (a) qpcr was used to assess the effects of mir- a and mir- mimics of mev genomic dna at the indicated times after h transfection with the mimics. data are from independent experiments (mean ± sd). statistical significance was analyzed by two-way anova test; *p < . ; **p < . ; ***p < . . (b,c,d,e) flow cytometric analysis was used to assess the effects of mir- a and mir- mimics on mev-infected f cells at the indicated times after h transfection with the mimics. the mean fluorescence intensities (mfi) of mev-infected f cells at the indicated times are shown. data are from independent experiments (mean ± sd). statistical significance was analyzed by student's t test; *p < . ; **p < . ; ***p < . . figure mir- a and mir- play roles on tfr and mev in a synergistic manner. (a) qpcr was used to assess effects of the mirnas on the relative expression of tfr after h transfection with the mimics, with β-actin as an internal control. (b) western blot assay was used to assess the tfr protein levels of lysates of f cells after h transfection with the mirnas mimics, with β-actin as an internal control. (c) qpcr was used to assess the effects of the mirnas on mev genomic dna at h post-mev infection, after h transfection with the mimics. data are from independent experiments (mean ± sd). statistical significance was analyzed by student's t test; *p < . ; **p < . . the viral genomic dna measured h later. as predicted, results showed that the two mirnas together produced a greater reduction in viral genomic dna than individually. host cellular mirnas have frequently been reported to interact with viruses during infection [ , , [ ] [ ] [ ] [ ] [ ] [ ] . we recently showed that mir- b inhibited mev replication by repression of its non-structural protein expression [ ] . here, we report that other mirnas, mir- a and mir- , inhibit mev entry into f cells by downregulating its receptor, tfr, through targeting the ′utr of tfr mrna. a number of reports have shown that tfr, as a cell surface receptor, is required for iron delivery to cells. indeed, tfr has been established as a gatekeeper for regulating iron uptake by most cells, and the transferring-to-cell endocytic pathway has been characterized in detail [ ] . tfr is central to the uptake of iron-loaded transferrin [ ] which is post-transcriptionally regulated via iron-responsive elements present in its ′utr [ ] . in addition to its role in erythropoiesis, tfr is also overexpressed in the majority of malignancies [ ] and is directly linked to cell proliferation [ ] [ ] [ ] . studies have demonstrated that parvovirus replication is dependent on host cellular division and proliferation ( [ ] ; tattersa [ ] ) and vigorous proliferative activity of host cells promotes parvoviral replication. reduction in cellular metabolic activity, therefore, may explain why downregulation of tfr inhibits mev replication. there may, however, be another mechanism. tfr is necessary for mev uptake by host cells: downregulation may therefore render infected cells less susceptible to superinfection with additional mev particles. while it may take only one virion to establish a primary infection, a multiply-infected cell may produce more infectious progeny. downregulation of tfr on the cell surface, therefore, may result in diminished production of virus. as summarized in figure , mev infection leads to increase in two host cell mirnas (mir- a and mir- ) which downregulate tfr expression, resulting in a decrease in viral replication. since tfr is a specific receptor of mev, however, reduction in it may prevent cellular entry of further virus following the primary infection, resulting in protection of host cells from immediate death as a result of continuing virus infection. although two host cell mirnas were found to be upregulated by mev infection, the mechanism remains unclear. we have identified one mechanism of interaction between mev and its host f cells, however, that might explain why a cell can be infected with just one virus. since mir- a and mir- have been shown to inhibit mev entry into host cells and might also affect virus replication, it may find application as an antiviral therapeutic for mev-induced mink enteritis. as several reports have shown [ ] [ ] [ ] [ ] [ ] , sirnas can be used to control virus diseases in vivo; however, little attention has so far been paid to the possibility of using endogenous mirnas as an antivirus tool. compared with sirnas, endogenous mirnas may be safer and induce fewer side-effects. more extensive studies are merited to determine if the two mirnas described here can be used as antiviral tools. in conclusion, our work has shown that two mirnas (mir- a and mir- ) inhibit mev entry into the f cells by downregulating its specific receptor tfr through targeting the ′utr of tfr mrna in a synergistic manner, while the two mirnas were upregulated through mev infection. as summarized in figure , a simple pathway of host-virus interaction network involving tfr and mirnas has been deduced. these results provide further understanding of the mechanisms in mev infection and may be helpful for development of endogenous mirna antiviral therapy strategies. two mink parvoviruses use different cellular receptors for entry into crfk cells the propagation of feline panleukopenia virus in microcarrier cell-culture and use of the inactivated virus in the protection of mink against viral-enteritis virus enteritis in mink. north amer vet studies on isolation, serum-free cultivation and manufacture of mink enteritis virus optimized for vaccine preparation analysis of the vp gene sequence of a new mutated mink enteritis parvovirus strain in pr china the functions of animal micrornas micrornas: genomics, biogenesis, mechanism, and function micrornas: genomics, biogenesis, mechanism, and function the widespread regulation of microrna biogenesis, function and decay epstein-barr virus latent membrane protein induces cellular microrna mir- a, a modulator of lymphocyte signaling pathways host-virus interaction: a new role for micrornas a viral microrna down-regulates multiple cell cycle genes through mrna ′ utrs epstein-barr virusencoded latent membrane protein (lmp ) induces the expression of the cellular microrna mir- a human cellular microrna hsa-mir- a interferes with viral nef protein expression and hiv- replication epstein-barr virus-encoded microrna mir-bart down-regulates the viral dna polymerase balf modulation of lmp a expression by a newly identified epstein-barr virus-encoded microrna mir-bart cellular micrornas inhibit replication of the h n influenza a virus in infected cells cellular microrna mir- b inhibits replication of mink enteritis virus by repression of non-structural protein translation binding site on the transferrin receptor for the parvovirus capsid and effects of altered affinity on cell uptake and infection combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains endocytosis and molecular sorting purified feline and canine transferrin receptors reveal complex interactions with the capsids of canine and feline parvoviruses that correspond to their host ranges canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells a microrna catalog of the developing chicken embryo identified by a deep sequencing approach initial sequence and comparative analysis of the cat genome genome annotation resource fields-garfield: a genome browser for felis catus soap: short oligonucleotide alignment program crispr recognition tool (crt): a tool for automatic detection of clustered regularly interspaced palindromic repeats rnahybrid: microrna target prediction easy, fast and flexible regrna: an integrated web server for identifying regulatory rna motifs and elements ifn induces mir- through a signal transducer and activator of transcription -dependent pathway as a suppressive negative feedback on ifn-induced apoptosis highthroughput real-time quantitative reverse transcription pcr microrna profile analysis of a feline kidney cell line before and after infection with mink enteritis virus hypersusceptibility to vesicular stomatitis virus infection in dicer -deficient mice is due to impaired mir and mir expression a liver-specific microrna binds to a highly conserved rna sequence of hepatitis b virus and negatively regulates viral gene expression and replication biological functions of micrornas: a review a cellular microrna mediates antiviral defense in human cells cellular microrna and p bodies modulate host-hiv- interactions interferon modulation of cellular micrornas as an antiviral mechanism transferrin receptor structure of the human transferrin receptor-transferrin complex the iron metabolism of neoplastic cells: alterations that facilitate proliferation? transferrin receptors in human-tissues -their distribution and possible clinical relevance regulation of transferrin receptor expression on human-leukemic cells during proliferation and induction of differentiation -effects of gallium and dimethylsulfoxide modulation of cell-surface iron transferrin receptors by cellular density and state of activation transferrin receptor expression and the control of cell-growth caillet-fauquet p: viruses and the cell cycle replication of the parvovirus mvm . dependence of virus multiplication and plaque-formation on cell-growth current prospects for rna interference-based therapies postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque an sirna-based microbicide protects mice from lethal herpes simplex virus infection protection against lethal influenza virus challenge by rna interference in vivo microrna mir- a and mir- inhibit mink enteritis virus infection by repression of its receptor, feline transferrin receptor the authors declare that they have no competing interests. key: cord- - t ss h authors: ali, mohsin; han, sangsu; gunst, chris j.; lim, steve; luinstra, kathy; smieja, marek title: throat and nasal swabs for molecular detection of respiratory viruses in acute pharyngitis date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: t ss h background: detection of specific respiratory viruses is important for surveillance programs, where nasopharyngeal or nasal swabs have traditionally been used. our objective was to determine whether sampling with a throat swab provides incremental benefit—when used in conjunction with a nasal swab—to detect respiratory viruses among patients with acute pharyngitis in the outpatient setting. findings: among university students with acute pharyngitis, we detected respiratory viruses with molecular assays on two samples collected per student: with a flocked nasal mid-turbinate swab and a rayon throat swab. forty-eight ( %) patients had virus-positive samples, with virus positives detected by either swab (one patient had a dual viral co-infection). the most common viruses were rhinovirus, coronavirus, and influenza a virus. specifically, virus positives were detected by both swabs, exclusively by the nasal swab, and six exclusively by the throat swab. the additional six virus positives detected by the throat swab corresponded to an absolute increase in viral detection of . % ( % ci: . – . %); the specific viruses detected were four rhinoviruses and two coronaviruses. conclusions: the flocked nasal swab samples respiratory viruses well, even among patients whose primary complaint is a sore throat. the rayon throat swab has modest incremental value over and above using the flocked nasal mid-turbinate swab alone, which suggests that while throat swabs alone would not be adequate for respiratory viral surveillance, they may have value as a supplementary test. acute pharyngitis is one of the most common illnesses for which patients visit primary-care physicians, accounting for - % of all visits [ , ] . for these patients, throat swabs are primarily used to detect streptococcus pyogenes, otherwise known as group a beta-hemolytic streptococcus (gabhs); however, most cases of acute pharyngitis are due to viruses. detection of specific viruses, especially influenza a, is important for respiratory viral surveillance programs, where nasopharyngeal or nasal swabs have traditionally been used. some recent research, however, suggests that incorporating throat swabs into these programs may increase sensitivity to detect respiratory viruses [ , ] . however, to our knowledge, no studies have quantified the incremental benefit that throat swabs could provide for surveillance of respiratory viruses. our primary objective was to determine whether sampling with a throat swab provides incremental benefit-when used in conjunction with a nasal swab-to detect respiratory viruses among adults with acute pharyngitis. we used a flocked mid-turbinate nasal swab, instead of a flocked nasopharyngeal swab, for several reasons. first, our study was done in an outpatient setting, where nasopharyngeal sampling for respiratory viruses does not routinely occur. in addition, previous research among adults has shown that flocked nasal mid-turbinate swabs are as sensitive for respiratory virus testing as flocked nasopharyngeal swabs, and since nasal mid-turbinate swabs are more comfortable and acceptable for patients, these swabs are preferred for sample collection in outpatient studies [ ] . there is also comparable sampling capacity between flocked nasopharyngeal and flocked mid-turbinate nasal swabs in terms of cell counts and levels of beta-actin, a measure of sampling adequacy [ ] . finally, nasal swabs are routinely used for the purpose of respiratory viral surveillance (the focus of our study), such as in the community setting for influenza. in terms of sample size, we specified a priori an absolute incremental benefit of % for viral detection (i.e., extra virus-positive samples detected by only the throat swab per patients) to be significant for the purpose of respiratory viral surveillance. to show this level of effect, we required patients, assuming a two-sided test, α = . and power of %. our secondary objective was to describe the viruses detected among patients with acute pharyngitis using molecular testing, which is not routinely done clinically. the study protocol was approved by the research ethics board at st. joseph's healthcare, hamilton, ontario, canada. from september through march , we recruited students presenting to mcmaster university's campus health clinic who were at least years old, reported a sore throat, presented within three days of symptom onset (to maximize viral detection) [ ] , and did not have concurrent serious illness, as judged by their health-care provider. after providing consent, patients completed a questionnaire regarding onset of illness and severity of signs and symptoms, adapted from a previously validated survey [ ] . trained study staff then sampled the patient's left side of their throat and tonsils with a rayon throat swab (copan italia); the left nostril was subsequently sampled using a flocked mid-turbinate nasal swab (floqswabs; copan italia). both swabs were placed into universal transport medium (utm; copan italia) and frozen at − °c until analysis. seven days later, patients were e-mailed a follow-up survey regarding the impact of their illness on their day-to-day lives. specimens were batch extracted using the nuclisens easymag assay (biomérieux canada; st. laurent, québec) and tested for respiratory viruses using the xtag respiratory virus panel (rvp) version (rvpv , luminex; austin, tx), which detects virus types and subtypes [ ] ; two laboratory-developed multiplex real-time polymerase chain reaction (pcr) assays for adenovirus [ ] , metapneumovirus [ ] , respiratory syncytial virus [ ] , and influenza a and b and parainfluenza - [ ] used by the hamilton regional laboratory medicine program for routine respiratory virus diagnosis; and a reverse-transcriptase pcr for enterovirus and rhinovirus [ ] . in addition, information regarding detection of betahemolytic streptococcal species (group a, c and g)-as diagnosed by the treating clinician during routine care-was also gathered per patient from the clinic, which did not use any of the specimens gathered from our study. as per standard of care, the clinic used rapid antigen detection tests for gabhs (rapid response strep-a, btnx; markham, ontario) and/or anaerobic culture on % sheep-blood agar plates followed by lancefield grouping (pathodx strep grouping, oxoid; nepean, ontario). furthermore, s. pyogenes was detected using the throat swabs collected in our study with pcr, using primers that have shown high sensitivity and specificity [ ] . eighty-three patients participated, of whom ( %) had a respiratory virus and/or beta-hemolytic streptococci detected in at least one sample. as shown in table , patients had virus-positive samples and had betahemolytic streptococci-positive samples ( gabhs and three group c); there were eight viral-streptococcal co-infections and one dual viral co-infection. the most common viruses detected were rhinovirus/enterovirus ( patients, of whom were confirmed as rhinovirus, and the remainder could not be resolved and were assumed to be rhinovirus), coronavirus ( patients), and influenza a ( patients). there were no salient differences regarding clinical presentation and one-week follow-up between patients detected with beta-hemolytic streptococci and various viruses ( table ) . of viruses detected with either the flocked midturbinate nasal or throat swabs, were detected by both swabs, were detected exclusively by the nasal swab, and six were detected exclusively by the throat swab, demonstrating that the flocked nasal swab eight patients had a viral-streptococcal co-infection. one patient had a viral co-infection of rhinovirus and coronavirus hku c beta-hemolytic streptococci were detected by rapid antigen detection (group a) and/or culture (group a, c, and g) outside our study, during routine clinical care. group a species were also detected within the study using pcr sampled viruses well even among patients whose primary complaint was a sore throat. results between the two swabs were concordant for % ( / ) of patients, with a moderate level of agreement (cohen's kappa, . ; % ci, . - . ). however, since rayon throat swabs detected six viruspositive patients who were negative in nasal swab samples, there was a small incremental value to its use, specifically, an absolute increase in viral detection of . % ( % ci, . - . %). the specific viruses detected were four rhinoviruses and two coronaviruses. this modest incremental benefit aligns with recent research on children and adults with influenza-like illness demonstrating that sampling the throat in conjunction with the nasopharynx or nose can improve sensitivity for viral detection [ , ] . this may be if the optimal site for detection (throat, nose, or nasopharynx) varies by virus, as shown in one previous study among patients with influenza-like illness and severe acute respiratory illness, which found higher sensitivity for detection for adenovirus and influenza a in the throat [ ] . therefore, sampling with throat swabs may be valuable in the context of respiratory viral surveillance, and a cost-effective approach could be to pool nasal and throat swabs together for combined processing and amplification [ , ] . our study has some limitations. without a control group of patients without acute pharyngitis, we could not distinguish infection unrelated to the presenting illness, and thus cannot infer causation. for example, it may be that the viruses detected in the nose did not cause pharyngitis. thus, our findings pertain more to the setting of respiratory viral surveillance, where the focus is on detection rather than diagnosis. second, the throat and nasal swabs we used were made of different materials (rayon vs. flocked nylon, respectively). some previous research suggests that flocked throat swabs may be superior to rayon throat swabs for respiratory viral detection. a study of newly admitted elderly inpatients in norway that tested oropharyngeal and nasopharyngeal specimens for respiratory viruses using real-time pcr found . -fold higher viral load ( % ci, . - , p = . ) with flocked swabs compared to rayon swabs, regardless of sampling site [ ] . this suggests that the incremental benefit we estimated for respiratory viral detection using the rayon throat swab may increase with a flocked throat swab. thus, swab material and other relevant factors regarding swab design-including swab length and shape-require further study. finally, as noted above, the optimal site for detection may vary by virus. we were unable to detect such differences due to our small sample size, and this also remains an area for future research. in summary, we evaluated rayon throat and flocked mid-turbinate nasal swabs for viral detection among young adults with acute pharyngitis. respiratory viruses were detected in the majority, particularly rhinovirus, coronavirus, and influenza a. the nasal swab sampled respiratory viruses well, even among patients whose primary complaint was a sore throat. we found modest incremental benefit for viral detection with the throat swab over and above the nasal swab alone, which suggests that while throat swabs alone would not be adequate for respiratory viral surveillance, they may have value as a supplementary test. acute pharyngitis outpatient visits for infectious diseases in the united states guidance for laboratory testing for detection and characterization of human influenza virus for the - respiratory virus season. manitoba: cphln comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing use of throat swabs or saliva specimens for detection of respiratory viruses in children the wisconsin upper respiratory symptom survey (wurss): a new research instrument for assessing the common cold development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay real-time pcr with an internal control for detection of all known human adenovirus serotypes evaluation of a new rapid antigen test using immunochromatography for detection of human metapneumovirus in comparison with real-time pcr assay simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus a and b by realtime pcr comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods rapid pcr detection of group a streptococcus from flocked throat swabs: a retrospective clinical study comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h n virus by real-time reverse transcriptase swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www this study was funded by st. joseph's healthcare, hamilton, canada. we thank santina castriciano and copan italia s.p.a. (brescia, italy) for contribution of all swabs and media; dr. jan young-baker, kathy patterson, rosanne kent and the nurses and doctors at mcmaster university's student wellness centre for support with study recruitment; kaitlyn boese for assistance in the laboratory; emma goodall for advice regarding the study protocol; brian a. chang, uttara partap and andrea smith for critical feedback on an earlier version of this manuscript; and to the students for their participation. includes patients with adenovirus (n = ), metapneumovirus (n = ) and respiratory syncytial virus (n = ) c includes patients with group a (n = ) and group c (n = ) beta-hemolytic streptococci, with five and three viral co-infections, respectively. no group g beta-hemolytic streptococci were detected d by definition, this proportion is % given study inclusion criteria the authors declare that they have no competing interests.authors' contributions ma and ms conceived and designed the study. ma, sh, cjg, and sl recruited patients, administered surveys, and collected study samples. kl carried out all laboratory assays. ma, sh, and ms analyzed and interpreted the data. ma and sh drafted the manuscript. all authors critically revised the manuscript and approved the final version. key: cord- -u cufg authors: finger, paula fonseca; pepe, michele soares; dummer, luana alves; magalhães, carolina georg; de castro, clarissa caetano; de oliveira hübner, silvia; leite, fábio pereira leivas; ritterbusch, giseli aparecida; esteves, paulo augusto; conceição, fabricio rochedo title: combined use of elisa and western blot with recombinant n protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: u cufg background: the avian infectious bronchitis virus (ibv) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. this study examined the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein (n) of ibv. the coding sequence for n was amplified by rt-pcr and expressed in escherichia coli. a soluble recombinant n protein (rn) of approximately kda was obtained. a total of sera were tested against the rn in elisa and the results were compared with those of the commercial idexx ibv ab test. elisa-rn achieved a . % sensitivity and . % specificity. wb confirmed all false negative sera in elisa-rn or idexx test as truly positive. the current study indicate that the combined use of rn in elisa and wb is a powerful tool for the immunodiagnosis of avian infectious bronchitis. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl (de ) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). a total of serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions ( : , : and : ) and different concentrations of purified rn antigen ( , and ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version . for windows (graphpad software, usa). results: the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately and kda. out of sera that scored positive in the commercial elisa idexx ibv ab test, were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of . %. out of sera that scored negative in the idexx ibv ab test, also scored negative in the elisa-rn, indicating a specificity of . %. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. methods: constructed recombinant pae/n expression vectors were used to transform e. coli bl (de ) star competent cells (invitrogen). the rn of infectious bronchitis virus was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). a total of serum samples from chickens were used to develop and evaluate the elisa-rn test. to standardize the indirect elisa development, serum dilutions ( : , : and : ) and different concentrations of purified rn antigen ( , and ng/well) were tested. positive and negative sera for ibv were used as controls. the results were compared with those obtained from a commercial kit. serum samples scored as negative with the commercial kit but as positive with the elisa-rn were further analysed by western blot analyses using the rn protein as an antigen. the results of the elisa-rn were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software graphpad prism version . for windows (graphpad software, usa). (continued on next page) (continued from previous page) results: the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to select for the coding region of the n protein. the rn was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately and kda. out of sera that scored positive in the commercial elisa idexx ibv ab test, were also positive in the elisa-rn, yielding an elisa-rn test sensitivity of . %. out of sera that scored negative in the idexx ibv ab test, also scored negative in the elisa-rn, indicating a specificity of . %. sera that tested negative in the elisa-rn and positive in the commercial test also reacted with the rn protein in western blot. conclusions: the association between the elisa and western blot techniques developed in this study with a subunit of ibv (rn) were able to detect antibodies that the commercial elisa did not detect suggesting that the elisa-rn has greater sensitivity. keywords: ibv, nucleoprotein, recombinant antigen, diagnosis, indirect elisa background avian infectious bronchitis (ib) is caused by a virus in the coronaviridae family, genera gammacoronavirus. it is a highly contagious disease with a short incubation period [ ] . the avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (ibv). the ibv is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms [ , ] . infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. secondary bacterial infections will also contribute to economic losses. carcass condemnation due to the development of airsacculitis [ , ] negatively impacts commercial sales of bird meat and eggs. brazil was once the world's largest exporter of poultry and currently the world's third largest producer of bird meat [ ] . the consequences of ibv are a significant threat to brazil's poultry industry. the ibv genome consists of a non-segmented positivesense single-stranded rna that is approximately . kb in length. it encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (n), the spike protein (s), the envelope protein (e), and the matrix protein (m). the nucleocapsid protein, or n protein, consists of amino acids. it has a molecular mass of approximately kda and directly binds with the viral genome to form the virion nucleocapsid [ , ] . its structure is highly conserved, with different strains of ibv sharing a high degree of identity ( - %) [ ] . the n protein is also known for its immunogenicity, inducing specific antibody and cytotoxic t-cells mediated responses [ , ] . there is significant interest in the use of the ibv n protein as an important target for diagnosis since it possesses the antigenic characteristics required for the development of serological assays that can be applied to detect or quantify antibodies against the ibv [ ] . the laboratory diagnosis of ib is dependent on direct and indirect techniques. the direct techniques are employed for viral isolation and genomic or phenotypic identification of the virus, while the indirect methods are used to detect specific antibodies [ ] . in addition to being applied for serodiagnosis, serological techniques can also be employed to evaluate the immune responses stimulated by vaccines. commercial elisa kits are typically used to indirectly diagnose ibv. these kits, however, are expensive when large number of samples require screening and they are not acessible for applications with the scale of the brazilian poultry industry [ ] [ ] [ ] . elisa techniques currently available are designed to detect polyclonal antibodies that target the whole virion. the use of nucleoprotein as the antigen for diagnosis and evaluation of vaccine immune responses is an interesting target to explore since this protein plays a important role in ibv virus replication and the induction of a specific immune response in infected birds [ , ] . the use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and, thereby, reduce or eliminate background reactions. the use of recombinant antigens also represents a viable method of reducing immunoassay development costs. easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis [ ] . the aim of the current study was to evaluate the combined use of an elisa and western blot (wb) to detect antibodies against the nucleocapsid protein of ibv. a previously characterized brazilian viral sample of ibv strain massachusetts (m -cnpsa -embrapa -concórdia, sc, brazil) was propagated after days of incubation in the chorioallantoic cavity of specific pathogen free (spf) embryonated chicken eggs. the allantoic fluid was then collected and stored at − °c. viral rna extraction was carried out with trizol® ls reagent (invitrogen™, eua), according to the manufacturer's instructions. extracted rna from ibv strain m was used for cdna synthesis with random oligonucleotides. reverse transcription (rt) was carried out using superscript® one-step rt-pcr system (invitrogen, usa). the resulting cdna samples were used to for pcr amplification of the whole orf of the n protein gene. primers based on the ibv m n protein gene sequence available at gen-bank (accession number m ) were designed to align between and and - bp of the gene and include cleavage sites for restriction enzymes. there was a restriction site for xhoi in the forward primer ( ′ -ccgctcgagatggcaagcggtaaggcaa - ′) and a restriction site for kpni in the reverse primer ( ′ -ggggtacctcaaagttcattctctccta - ′). the pcr reaction was performed with approximately ng of the extracted cdna, . mm mgcl, . mm dntps, units of taq dna polymerase, x reaction buffer, pmol of each primer, and m n,n,n-trimethylglycine (betaine) under the following conditions: cycle of °c for min, cycle of °c for min, then cycles of °c for min, °c for min, and °c for min, and a final extension of °c for min. the pcr amplification product was confirmed on a % agarose gel and purified using gfx pcr dna and gel band purification kit (ge healthcare, chicago, usa), according to the manufacturer's instructions. the pcr product was cloned into pae vectors by a t dna ligase (invitrogen) binding reaction after cleavage with restriction enzymes kpni and xhoi. the constructed recombinant pae/n expression vector was used to transform e. coli bl (de ) star competent cells (invitrogen). the resulting recombinant clones were cultivated in ml of lb broth medium with μg/ml of ampicillin ( °c, h, rpm). the whole culture volume was transferred to flasks containing ml of lb and incubated at °c with agitation ( rpm) until the optical density (o.d.) at nm reached . . the expression of the recombinant n protein (rn) was induced by adding isopropyl-β-d-thiogalactopyranoside (iptg, . mm final concentration) to the culture and incubating for h at °c with agitation ( rpm). the cells were harvested at , x g for min at °c and the culture pellet containing the rn protein was subjected to a solubilization procedure in Äkta wash buffer ( . % nah po , . % of nacl, . % imidazole, ph . , supplemented with μg/ml lysozyme). the resuspended cells were submitted to seven sonication cycles of s at hz and centrifuged again. the rn was purified by affinity chromatography using histrap hp ml columns pre-packed with pre-charged ni sepharose in the Äktaprime automated liquid chromatography system (ge healthcare). the protein concentration was determined with qubit™ protein assay kits (thermo-fisher scientific, usa) according to the manufacture's instructions. the sds-page was carried out on % polyacrylamide gel. the gels were either stained with coomassie blue r- (bio-rad, california, usa) or electroblotted onto hybond-ecl . μm nitrocellulose membranes (ge healthcare) using the bio-rad mini trans-blot cell (bio-rad) for western blot. briefly, the membrane was blocked with % non-fat milk in phosphate buffer saline containing . % tween- (pbs-t) ( mm nacl, . mm kcl, mm na hpo , mm kh po , ph . ), incubated with mouse monoclonal antibody (mab) anti- xhis (sigma, usa) diluted to : in pbs-t, and incubated with polyclonal antibody anti-mouse igg conjugated to hrp (sigma). all incubation steps were performed at °c for h under slight agitation followed by three washes with pbs-t. the immunoblot was developed using , ′-diaminobenzidine (sigma). a total of chicken serum samples (n = ) were used to develop and evaluate the elisa-rn. the serum samples were kindly provided by mercolab laboratories (garibaldi, brazil), a laboratory accredited by the mapa (ministry of agriculture, livestock and food supply) that uses the commercial elisa kit ibv ab test (idexx) for characterization. serum samples from chickens with a positive diagnosis for newcastle disease were used for the evaluation of the elisa-rn specificity. the samples used in this study originated from birds with a known history of vaccination, including vaccination against the newcastle disease virus, and were categorized into three groups: commercial egg-layers, broiler breeders, and meat-producing chickens. this information was further used to improve the evaluation of the elisa-rn test. were tested. positive and negative sera for ibv were used as controls. the -well microtiter plates (nunc max-isorp®, thermo fisher, usa) were coated with ng/ well of rn diluted in . m carbonate-bicarbonate buffer (ph . ) and incubated overnight at °c. the plates were incubated at °c for h with a blocking solution ( % non fat dry milk in pbs-t). serum samples diluted : in pbs-t were added in duplicate and the plates were again incubated for h at °c. after this period, the secondary antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) diluted : , in pbs-t was added and plates were once more incubated at °c for . h. after each incubation, the plates were washed three times with pbs-t. in the final step, plates were washed five times and μl/well of peroxidase substrate o-phenylenediamine dihydrochloride (sigma aldrich) was added. the reaction was interrupted with n h so and the results were read as o.d. using a spectrophotometer at nm. to determine the cutoff value, negative sera were used, totalizing sera, considering the mean of sera added of two standard deviation. the roc analysis was used to simulate the influence of different cutoff values on the sensitivity and specificity of the test. the results were compared with those obtained with the commercial kit. serum that scored as negative in the commercial kit but as positive in the elisa-rn were submitted to western blot analyses using the rn protein as antigen. in order to evaluate the repeatability of the elisa-rn, three separate batches of recombinant n protein were produced and purified following the methodology described above with distinct purification times, to demonstrate the reproducibility of the recombinant protein expression procedure. serum samples were selected for testing against each batch of antigen and the averages, and standard deviations, of the o.d. at nm were calculated. of the samples used for this test, one was strongly reactive positive, four were moderately reactive positive, and four were negative serum samples. to evaluate the specificity of the test, negative serum samples to ibv and positive serum samples to newcastle disease were tested in the elisa-rn. the western blot was performed almost as described above. the rn was deposited in all wells of the sds-page gel and, after transfer to the nitrocellulose membrane, it was cut to obtain strips. each strip was incubated with a : serum dilution after blocking with % non-fat milk. the antibody horseradish peroxidase (hrp) -rabbit anti-chicken igy peroxidase conjugate (sigma) ( : ) was added to all membranes and the reaction was revealed using , ′-diaminobenzidine (sigma). all incubation steps were performed at °c for h under slight agitation and were followed by three washes with pbs-t. the results of the elisa-rn compared to the commercial kit results, the receiver-operating characteristics (roc) curves, the area under the curve (auc) and the confidence intervals were obtained through the software graphpad prism version . for windows (graphpad software). the expected cdna fragment of approximately bp was successfully amplified by pcr using primers designed to obtain the coding region of the n protein. successful recombinant pae/n vector construction was confirmed through cleavage with the same restriction enzymes used for the plasmid construction (date not shown). the recombinant n protein (rn) was expressed as a soluble protein, dispensing refolding steps, and, after purification, a yield of mg/l of rn was obtained. the sds-page results demonstrated the presence of two distinct bands that had a molecular mass of approximately kda and kda (fig. a) . the same bands were observed in the western blot and confirmed the presence of bands that corresponded to the protein of interest (fig. b) . other studies have also described the cloning of the bp corresponding to the n protein gene from ibv [ , ] . they reported that the recombinant protein presented as two distinct molecular masses, one kda and other kda. the kda mass is the truncated form of the n protein [ , ] . this corroborates with the results observed through sds-page and western blot in the current study (fig. ) . the results of the elisa-rn were compared with the results obtained with the ibv ab test (idexx). receiver-operating characteristics (roc) with % confidence intervals were used to analyse these results (fig. ) . out of sera that scored positive in the ibv ab test, also scored positive in the elisa-rn test. the sensitivity of the elisa-rn test was . %. out of the sera that scored negative in the idexx test, also scored negative in the elisa-rn, indicating a specificity of . %. the area under the curve (auc) was . (p < . and cutoff . ). three different protein batches were used as coating antigens for testing the elisa-rn and the results from all three were similar (p > . ) and, thus, indicate antigen stability (fig. ) . all newcastle disease positive sera tested negative in the developed elisa-rn, providing evidence of test specificity (data not shown). all sera that tested negative in the commercial test and positive in the elisa-rn were submitted for western blot analyses using the rn protein as an antigen. all serum samples reacted with the two bands of rn, confirming the results obtained in the developed elisa (fig. ) . sera that tested negative in the elisa-rn and previous studies that focused on the development of more efficient diagnostic techniques were not limited to avian infectious bronchitis detection, but also aimed to control and monitor chickens vaccination [ , ] . the nucleoprotein from ibv is widely considered the choice protein for the development of immunoassays for antibody detection since this protein plays an important role in inducing an antibody response in ibv infected or vaccinated animals [ , ] . other studies have demonstrated that the nucleocapsid protein is highly conserved among ibv isolates ( - . % similarity), immunogenic, and abundantly expressed during infection [ , , ] . these features make this protein an interesting candidate for use in diagnostic techniques, such as elisa and wb [ ] . the elisa is a powerful tool because it provides a safe and an easy way to evaluate the ibv vaccine efficiency, and perform serological diagnosis as well as epidemiological surveillance [ ] . in contrast to the elisa presented in this study, the elisa developed by lugovskaya et al. [ ] used two fragments of the recombinant n protein expressed in e. coli as an antigen, achieved a specificity of . %, and a sensitivity of . %. these results are comparable with the commercial test that is currently used for routine ibv diagnosis that has a specificity of . % and a sensitivity of . % [ ] . the elisa-rn developed in this study presented similar specificity and sensitivity results ( . and . %, respectively), and thus highlights that our study uses a western blot technique to complement the results obtained through elisa and further support the ibv diagnosis. the western blot technique may prove useful for epidemiological studies, for monitoring specific pathogen free farms, and in vaccine potency tests. the association of elisa and western blot can be seen as a tool to be considered to increase sensitivity for serodiagnosis purposes [ ] . since positive sera react with two bands in the western blot, the technique is even more specific. the recombinant n protein produced in this study was expressed in the soluble fraction. our rn was easily recoverable from the culture without using denaturing agents. in contrast to previous studies where the n protein was expressed in its insoluble form [ , ] , our procedure avoids the necessity to refold the recombinant protein. the production of rn was repeatable since the same elisa-rn test results, and about the same yield, were obtained when different production and purification times were used. besides the use of the n protein for the diagnosis of ibv, an elisa that was developed using the s protein was also described and compared to a commercial test [ ] , and achieved a specificity and sensitivity of . and . %, respectively. however, the n protein offers specific advantages when used for the purpose of diagnosis during viral infection since it is produced in larger quantities than the s protein (the n proteins is produced at a ratio of : relative to the s protein [ ] and plays an important role in the virus's replications and assembly process [ ] . additionally, the s protein has hypervariable regions that cause mutations in its sequence and therefore offers low efficiency as a protein for diagnosis [ ] . the results from the elisa test developed using the rn protein indicate that the test could be effectively applied for ibv diagnosis [ ] . the yield of rn per litre of lb broth culture would coat approximately -well microtiter plates allowing for the diagnostic analysis of approximately , serum samples in duplicate. also noteworthy is the production cost reduction from using rn in a soluble form, since this eliminates costs associated with the refolding step while it also preserves important conformational epitopes. the currently available commercial test on the other hand, employs the whole ibv as an antigen which requires viral propagation and the implementation of robust laboratory biosafety standards [ ] . it is worth mentioning that, by comparing the developed elisa-rn and wb with the idexx ibv ab test, the analysis conducted in the current study indicated that it was possible that some serum identified as negative for anti-ibv antibodies on the commercial test be positive when tested using the elisa-rn and by wb. this could be of concern as false negative results are undesirable, especially if a lot of birds are misdiagnosed. thus, the elisa-rn could be applied together with the wb developed in this study for the routine detection of ibv in diagnostic laboratories. false negative results that contribute to the spread of the disease can be avoided, or at least decreased, when the two tests are applied together. the indirect elisa developed here with rn as an antigen allowed for the detection of anti-ibv antibodies in chicken serum at high specificity and sensitivity. the association between elisa and western blot techniques developed with a subunit of ibv (rn) were able to detect antibodies that were not detected with the commercial elisa test suggesting greater sensitivity in the developed elisa-rn. in addition, the elisa-rn with the advantages of easy preparation and improved safety could be a promising alternative to the whole live virus elisa. relationship between sequence variation in the s spike protein of infectious bronchitis virus and the extent of crossprotection in vivo infectious bronchitis virus: immunopathogenesis of infection in the chicken efficacy of infectious bronchitis virus vaccines against heterologous challenge coronavirus avian infectious bronchitis virus bronquite infecciosa das galinhas: conhecimentos atuais, cepas e vacinas no brasil polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses comparisons of the structural proteins of avian infectious bronchitis virus as determined by western blot analysis specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in brazil recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry high-level protein expression following single and dual gene cloning of infectious bronchitis virus n and s genes using baculovirus systems detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay the s glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens selective replication of coronavirus genomes that express nucleocapsid protein elisa for antibodies to infectious bronchitis virus based on nucleocapsid protein produced in escherichia coli evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus diversidade genética de amostras brasileiras do vírus da bronquite infecciosa determinada pelo sequenciamento de nucleotídeos dos genes n e s development of a recombinant nucleoprotein-based enzyme-linked immunosorbent assay for quantification of antibodies against porcine reproductive and respiratory syndrome virus combined use of western blot / elisa to improve the serological diagnosis of human tuberculosis development of a multiepitope antigen of s protein-based elisa for antibodies detection against infectious bronchitis virus the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with ′ genomic rna coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides we thank mercolab laboratory (garibaldi, rs, brazil) for giving the sera that were analyzed in this study. not applicable. all data generated or analyzed during this study are included in this article. all authors read and approved the final manuscript. this article does not contain any studies with human participants or animals performed by the author. the authors authorize the publication. no known competing interest. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -otc pc authors: liu, xiaoli; han, zongxi; shao, yuhao; yu, dan; li, huixin; wang, yu; kong, xiangang; liu, shengwang title: identification of a novel linear b-cell epitope in the ul and ul . proteins of duck enteritis virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: otc pc background: the unique long (ul ) and ul . proteins of herpes simplex virus are known to function during the assembly of the viruses. however, for duck enteritis virus (dev), which is an unassigned member of the family herpesviridae, little information is available about the function of the two proteins. in this study, the c-terminus of dev ul protein (designated ul c), which contains the whole of ul . , was expressed, and the recombinant ul c protein was used to immunize balb/c mice to generate monoclonal antibodies (mab). the mab c was generated against dev ul and ul . proteins and used subsequently to map the epitope in this region. both the mab and its defined epitope will provide potential tools for further study of dev. results: a mab (designated c ) was generated against the dev ul c protein, and a series of partially overlapping fragments that spanned the dev ul c were expressed with gst tags. these peptides were subjected to enzyme-linked immunosorbent assay (elisa) and western blotting analysis using mab c to identify the epitope. a linear motif, ( )iyypge( ), which was located at the c-terminus of the dev ul and ul . proteins, was identified by mab c . the result of the elisa showed that this epitope could be recognized by dev-positive serum from mice. the ( )iyypge( )motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of c with several truncated peptides derived from the motif. alignment and comparison of the c -defined epitope sequence with those of other alphaherpesviruses indicated that the motif ( )yypge( )in the epitope sequence was conserved among the alphaherpesviruses. conclusion: a mab, c , was generated against dev ul c and the epitope-defined minimal sequence obtained using mab c was ( )iyypge( ). the mab and the identified epitope may be useful for further study of the design of diagnostic reagents for dev. herpesviruses exist widely in nature. the genomes of herpesviruses consist of linear double-stranded dna; they differ in size (from approximately to kb), sequence arrangement and base composition [ ] , and vary significantly with respect to the presence and arrangement of inverted and directly repeated sequences. the genomes of most of the alphaherpesviruses, such as herpes simplex virus (hsv- ) [ ] and marek's disease virus (mdv- ) [ ] , encode more than proteins; some of these proteins are not essential for the replication of the viruses. only limited information is available about the structures and functions of these proteins, although some studies of the antigenic determinants of the glycoproteins have been reported [ , ] . three types of capsid, named a-, b-, and c-capsids, are needed in the assembly of hsv- [ ] . b-capsids lack dna but may be the important intermediates in virus assembly [ ] [ ] [ ] [ ] . the unique feature of b-capsids is the presence of an abundant core protein, named scaffolding protein icp (vp a) [ , [ ] [ ] [ ] , which is encoded by the in-frame gene ul . . this protein is present in the b-capsids of the hsv- assembly but is absent after the completion of dna encapsidation and is not found in the mature virion [ ] . duck enteritis virus (dev), an unassigned member of the family herpesviridae [ ] , is the cause of duck viral enteritis (dve), which is also known as duck plague (dp), a disease of anseriformes. dve is a form of hemorrhagic enteritis that occurs in captive or free-flying waterfowl [ ] and causes heavy economic losses in commercial duck production [ ] . the dev establishes an asymptomatic carrier state in waterfowl in the course of infection, and it is only detectable during the intermittent shedding period of the infection [ ] . currently, only limited information is available on the genomic sequence and encoded proteins of dev; therefore the development of diagnostic methods based on virus detection is difficult. hence, the development of immunity based prophylactic, therapeutic, and diagnostic techniques for the control dev is of significance. the dev has a linear double-stranded dna genome of approximately kb with a g+c content of . % [ ] . the genes and their arrangements in the dev ul region have been reported by our laboratory [ ] [ ] [ ] [ ] [ ] . our results have demonstrated that dev ul and ul . , two nested in-frame genes, encode a capsid maturation protease and the minor capsid scaffold protein of dev [ ] . b-cell epitopes are antigenic determinants that are recognized and bound by membrane-associated receptors on the surface of b lymphocytes [ ] . they can be classified into two types: linear (continuous) epitopes and conformational (discontinuous) epitopes. linear epitopes are short peptides that correspond to a contiguous amino acid sequence within a protein [ , ] . to date, there has been no study of the b-cell epitopes of dev. in this study, we first expressed the amino acids in the c-terminus of the dev ul protein (named ul c), which contain the whole sequence of ul . . subsequently, we generated a monoclonal antibody (mab) (named c ) against dev ul by vaccination of mice with a recombinant ul c prime and dev particle boost. finally, we identified an epitope in the dev ul protein which was recognized by the mab c . these results will provide a basic understanding of the structure, function and localization of the dev ul protein and ul . protein. this mab and the recombinant proteins could be used as a potential tool for the design of diagnostic reagents for dev. owing to the difficulty of expressing the whole ul gene (in total bp in length) of dev, we used prokaryotic expression of the c-terminal -amino acid ( - aa) protein in e. coli bl (de ) after induction by isopropyl-β-d-thiogalactopyranoside (iptg) and termed the recombinant protein ul c. western blotting using murine antibody against dev showed that ul c could react with dev antibody ( figure a ), which implied that it had similar antigenicity to native dev ul protein. the ul c was used to prime immunity and dev particles were used as boost antigens to prepare the mab. one hybridoma clone that secreted mab specific to the dev ul protein was selected and designated as c . the subclass of c was determined to be igm with light chain. both western blotting and elisa showed that c could react specifically with both chicken embryonic fibroblasts (cef) infected with dev ( figure b and figure c ) and the recombinant protein ul c ( figure d and figure c ). in summary, we can conclude that the mab c recognized the dev ul protein specifically. for fine mapping of the epitope of dev ul that was recognized by mab c , a set of gst fused proteins ( figure ) were expressed in prokaryotes and used together to identify the epitope by both western blotting and elisa. western blotting showed that the peptide f ( - aa) was recognized by mab c ( figure a ). the further screening results showed that both f - ( - aa) and f - ( - aa) could react with mab c , while f - ( - aa) failed to be recognized ( figure a ). these results indicated that the overlapping sequence shared by f - and f - may contain the epitope recognized by mab c . hence, to define the epitope defined by mab c in the ul protein more precisely, a series of truncated peptides (from f to f ; figure ) that were deleted successively from the amino or carboxyl terminuses of the overlapping fragment were expressed, respectively, for subsequent screening of mab c . the results showed that the mab c reacted with peptides from f ( - aa) to f ( - aa) ( figure b and figure c ), but not with f ( - aa) and f ( - aa) ( figure c ). therefore, we considered that the motif iyypge is the defined minimal epitope in the ul protein of the dev clone- strain that is recognized by mab c , because deletion of i or e destroyed the binding of the gst fusion peptides by mab c . in addition, the results were confirmed further by elisa ( figure d ). the recombinant peptide, f ( iyypge ), was used as an antigen to react with mouse anti-dev antibody in this study. the results of both western blotting and elisa showed that this peptide defined by mab c could react with murine anti-dev antibody ( figure ), which demonstrated that the epitope had good reactivity. the mab c -defined motif and flanking sequences in the ul protein of alphaherpesviruses were aligned and analyzed. the results showed that the epitope defined by mab c , iyypge , was relatively conserved in alphaherpesviruses ( figure ). interestingly, ehv- and ehv- have the same amino acid residues as dev in the motif defined by mab c . two residues of the epitope, yy , were highly conserved among the alphaherpesviruses, except for the residue f in vzv. in addition, the c-terminus of the amino acids of the epitope was highly conserved among most of the alphaherpesviruses selected for this study ( figure ). formation of the herpesvirus capsid is the first step in viral morphogenesis. the capsid of hsv- is found in the mature virions and in the nuclei of infected cells from which they originate. there are three distinct types of capsid, a-, b-and c-capsids, in infected cells [ ] . the b-capsids of hsv- contain a large amount of the scaffolding protein (the product of the ul . gene), and smaller amounts of vp and vp , the products of the ul gene. the ul and ul . genes are expressed as '-coterminal transcripts, and the promoter for the ul . gene is located within the coding region of the ul gene in hsv- [ ] [ ] [ ] . the ul gene encodes a self-cleaved protease that generates the capsid proteins vp and vp [ ] . cleavage of the ul and ul . proteins is not required for capsid assembly, but the cleavage event is essential for dna encapsidation [ ] . the ul . gene encodes the scaffold protein vp a [ ] , which has been linked to a family of proteins named infected-cell protein (icp ). the vp a is the most abundant protein found in the b-capsid and may form the scaffold of the hsv- b-capsid [ ] . the b-capsid is similar to the capsid found in infectious hsv- particles, except that it lacks dna. the cavity of the b-capsid is occupied instead by a proteinaceous core table . the bars represent peptides of the truncated dev ul proteins. the peptides that were negative in western blotting and elisa with mab c are shown in gray and the peptides that were positive in western blotting and elisa with mab c are shown in pink. that is removed when dna enters [ ] . the ul proteinase cleaves the products of the ul . gene, and itself, at a site amino acids from the c terminus of its product [ ] . dev is an unassigned member in the family herpesviridae and, to date, there has been no report of the structure and function of proteins ul and ul . in dev. it has been reported that the genes ul and ul . in dev are similar to their homologues in other alphaherpesviruses [ ] . it has been speculated that they have similar functions in viral infection and replication to those of hsv- and other alphaherpesviruses. in this study, we expressed the c-terminus of dev ul and immunized mice with both recombinant protein ul c and the dev particles, using a prime-boost protocol, and one mab ( c ) was generated by cell fusion. interestingly, six bands were detected in devinfected cefs in western blotting analysis using mab c in this study ( figure b) . these may have originated from cleavage by the products of the ul gene. figure shows the proteins that are predicted to originate from the products of the dev ul and ul . by the alignment of the homologous in hsv- . the dev ul gene encodes a deduced protein of amino acids [ ] . comparison of the amino acids in dev ul with the homologous in hsv- showed that dev ul contains two sites. the release site (r) was at amino acids and , and the maturational site (m) was at amino acids and . self-cleavage by ul may yield the proteolytically active vp -like protein and vp -like protein ( figure ) [ ] , which are approximately kd and kd, respectively. the ul . gene is in frame with ul ; therefore, the ul . -encoded proteins possess the same m site as the figure reactivity of the identified epitope (f : iyypge ) with antibodies against dev. a) the peptide that corresponded to the epitope defined by mab c was used as the coating antigen in an elisa, and purified rgst protein was used as a negative control. the murine anti-dev antibody was used as the primary antibody and spf mouse sera were used as negative antibody controls. b) the western blotting results of the epitope defined by mab c with the murine antibody against dev; the rgst and e. coli bl (dh ) without induction were used as negative controls. ul protein. consequently, we hypothesized that the six bands detected using western blotting in this study may indicate the six products of ul and ul . . a series of fragments that spanned the ul c protein were expressed with a gst tag in this study, and used to screen for the minimal epitope recognized by mab c using western blotting and elisa. it was demonstrated that the minimal sequence of the epitope defined by mab c appeared to be iyypge , because any deletion of residues from either end of iyypge destroyed the ability of mab c to bind. comparative analysis of the amino acid sequences of the identified epitope with those of another alphaherpesviruses revealed that the c-terminus of the linear b-cell epitope yypge is conserved among the selected alphaherpesviruses, except prv, vzv and iltv. it has been reported previously that the motif yypge is conserved in the scaffold proteins of alphaherpesviruses [ ] . it was reported that deletion of a amino acid motif from the hsv- ul . , comprising amino acids to , which contained the sequence yypge, did not affect the formation of capsids in vitro but had a specific effect on incorporation of the portal. this indicated that this deletion had blocked dna packing but had not interfered with the assembly of bcapsids [ , ] . therefore, the -amino acid motif that contains yypge is required for the formation of portalcontaining capsids in hsv- -infected cells, and it is essential for the production of infectious virus [ ] . however, in dev, the function of the motif needs further investigation. the result of the in vitro neutralization test showed that mab c could not neutralize the infectivity of dev. the absence of neutralizing activity against dev might indicate that this region has low immunogenicity or, more probably, that this region is not exposed on the surface of the virion. indeed, the products of the hsv- ul gene are components of viral capsids, which are located inside of the viral tegument and envelope, while the products of the ul . gene are components of b-capsids [ ] . the products encoded by the two genes show predominantly nuclear localization within infected cells [ ] . the b-capsids are accumulated in the nucleus prior to viral dna encapsidation [ ] and the predominant component of b-capsids is the product of the ul . gene, which also contains the sequences of the epitope defined by c . the mab c and its epitope, which was defined in this study, may prove to be very useful tools for the development of immunity-based therapeutic and diagnostic techniques for dev, although the mab lacked neutralizing ability. in this study, we generated a mab, c , and identified a novel linear b-cell epitope on the dev ul and ul . proteins using this mab. the identified epitope and mab c may increase our understanding of the function and location of the ul and ul . proteins in dev. it will also be a potential tool for the design of diagnostic reagents for dev. the sp / cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen, usa) supplemented with % fetal bovine serum (fbs) (invitrogen, usa) and % penicillin-streptomycin in a humidified % co atmosphere at °c. chicken embryo fibroblasts (cef) were prepared from -to -day-old specificpathogen-free (spf) embryonated eggs according to standard procedures. the dev clone- strain was purified from a commercial chinese dev vaccine using the plaque assay, as described previously [ ] . the virus was propagated in cef in dmem with % fbs and harvested when the cytopathic effect (cpe) reached %. after three freeze-thaw cycles, cell lysates were confirmed primarily by electron microscopy (jem- ex, japan) and polymerase chain reaction (pcr) [ ] . given that it is difficult to induce prokaryotic expression of the full ul gene, we expressed a fragment of bp at the c-terminus of the dev ul gene (ul c) by the results were based on the alignment of the structure of ul in dev and hsv- [ , ] . the sequence of the epitope is indicated by the yellow bars. each of the proteins is designated by ul or ul . plus the sites of cleavage. the pink bars represent the amino acid sequence that contains the epitope defined by mab c . the gray bars represent the proteins that were not recognized by mab c . amplifying the gene fragment from dev clone- . the sequences and locations of the primers used for amplification and expression of the gene in this study are shown in table . after construction, each recombinant expression construct was transformed into e. coli bl (de ) (novagen, gibbstown, nj, usa). a series of fusion proteins with the expected molecular weights were induced by iptg and stained with coomassie blue after sds-page as described previously [ ] . for preparation of purified proteins, inclusion body proteins were separated by sds-page, the proteins of interest were excised, and the gel slices were crushed and added to an appropriate volume of sterilized pbs. the extracted proteins were used for western blotting and elisa. three -week-old balb/c mice were primed subcutaneously with dev virus particles mixed with an equal volume of freund's complete adjuvant (sigma, usa), followed by two boosts of immunization with the recombinant protein ul c. the protocols used for the preparation of mabs and ascitic fluid have been described previously [ , ] . all hybridomas were cloned by at least three rounds of limiting dilution. the class and subclass of the mab produced were determined using an sba clonotyping™ system/hrp kit (southern biotechnology associates, birmingham, usa). the specificity and reactivity of the mab c were also determined by western blotting using recombinant ul c protein and cef infected by dev, respectively. purified recombinant ul c protein, truncated proteins and dev in cef were separated by denaturing sds-page. for western blotting, all the proteins and the virus were transferred onto nitrocellulose membranes, and detected as described previously [ ] . purified recombinant gst (rgst) protein and the cef were used as negative antigen controls. the reactivity of the mab with different truncated recombinant ul proteins was determined further by elisa as described previously [ ] . briefly, the purified recombinant proteins were used as coating antigens, applied at μg/well in . m carbonate buffer (ph . ) at °c for h, and blocked with . % bsa at °c for h. after washing three times with pbst, μl of mab were added to the wells and incubated at °c for h. the plates were washed three times and incubated with hrp-conjugated sheep anti-mouse secondary antibody at °c for h. the color was developed and the reaction terminated, and the absorbance was measured at nm. all assays were performed in triplicate. the mab c was tested for the presence of dev-neutralizing antibodies. the cef lysates containing dev were mixed with ascites fluid containing mab c and incubated at °c for h; unrelated ascites fluid and pbs, used as negative controls, were treated in the same way. the mixture was added to the prepared cef and incubated at °c. after h of incubation, the mixture was removed and the cells were overlaid with % low-melting-point agarose containing % fbs. at h post-incubation, the cells were overlaid again with % low-melting-point agarose containing . % ponceau. after further incubation at °c for h, the plaques were counted and compared. detection of the reactivity of the epitope defined by mab c to investigate whether the peptides could be recognized by anti-dev antibody, the epitope peptide f was purified and used as antigen to coat elisa plates ( μg/ well) to react with mouse anti-dev antibody and mouse sera, respectively. purified rgst was used as the negative control for f . in addition, the purified f and rgst were also used to detect the reactivity of the epitope peptide by western blotting. homologous analysis of the sequence of the epitope defined by mab c the mab c -defined epitope sequences and flanking sequences of dev were compared with those of other selected herpesviruses of the alphaherpesvirinae using the megalign program in lasergene (dnastar) with clustal w multiple alignments, as described previously [ ] . the herpesviruses used in this study were as follows: equine herpesvirus (ehv- ) genomic organization and evolution of the human herpesviruses the genome sequence of herpes simplex virus type the genome of a very virulent marek's diseases virus distributions of bcell epitopes on the pseudorabies virus glycoprotein b identification of another b-cell epitope in the type-specific region of equine herpesvirus glycoprotein g proteins specified by herpes simplex virus viii. characterization and composition of multiple capsid forms of subtypes and characterization of three species of equine herpesvirus (ehv- ) biochemical studies of the maturation of herpes virus nucleocapsid species identification and characterization of a herpes simplex virus gene product required for encapsidation of virus dna characterization of intranuclear capsids made by ts morphogenic mutants of hsv- use of ar plasmid etching to localize structural proteins in the capsid of herpes simplex virus type structure of the herpes simplex virus capsid: effects of extraction with quanidine hydrochloride and partial reconstitution of extracted capsids structure of the herpes simplex virus capsid molecular composition of the pentons and triplexes capsid assembly and dna packing in herpes simplex virus the order herpesvirales molecular characterization of the dna of anatid herpesvirus duck plague on the american continent duck plague: a carrier state in waterfowl characterization of the genes encoding ul , tk and gh proteins from duck enteritis virus (dev): a proof for the classification of dev molecular characterization of the herpes simplex virus (hsv- ) homologues, ul to ul , in duck enteritis virus (dev) the ul to ul gene sequences of duck enteritis virus correspond to their homologs in herpes simplex virus comparative analysis of the genes ul through ul of the duck enteritis virus and other herpesviruses of the subfamily alphaherpesvirinae unique sequence characteristics of genes in the leftmost in the leftmost region of unique long region in duck enteritis virus identification of epitope-like consensus motifs using mrna display continuous and discontinuous protein antigenic determinants characterization of a protective linear b cell epitope against feline parvoviruses the herpes simplex virus gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate characterization of the protease and other products of amino-terminus-proximal cleavage of the herpes simplex virus ul protein the size and symmetry of b capsids of herpes simplex virus type are determined by the gene products of the ul open reading frame identification of the herpes simplex virus- protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products the protease of herpes simplex virus type is essential for functional capsid formation and viral growth assembly of herpes simplex virus (hsv) intermediate capsids in insect cells infected with recombinant baculoviruses expressing hsv capsid proteins identification of a region in the herpes simplex virus scaffolding protein required for interaction with the portal amino acids - of the herpes simplex virus scaffold protein are required for the formation of portal-containing capsids open reading frame ul of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain autoproteolysis of herpes simplex virus type protease release an active catalytic domain found in intermediate capsid particles identification of a novel linear b-cell epitope in the m protein of avian infectious bronchititis coronaviruses continuous cultures of fused cells secreting antibody of predefined specificity the complete dna sequence of the long unique region in the genome of herpes simplex virus type identification of a novel linear b-cell epitope in the ul and ul . proteins of duck enteritis virus authors' contributions xl, sl and xk designed research; xl, zh, ys and dy performed research; xl, sl, xk, hl and yw analyzed data; and xl, sl and xk wrote the paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - njwigco authors: maher-sturgess, sheryl l; forrester, naomi l; wayper, paul j; gould, ernest a; hall, roy a; barnard, ross t; gibbs, mark j title: universal primers that amplify rna from all three flavivirus subgroups date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: njwigco background: species within the flavivirus genus pose public health problems around the world. increasing cases of dengue and japanese encephalitis virus in asia, frequent outbreaks of yellow fever virus in africa and south america, and the ongoing spread of west nile virus throughout the americas, show the geographical burden of flavivirus diseases. flavivirus infections are often indistinct from and confused with other febrile illnesses. here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups. results: bioinformatic analysis of published full-length flavivirus genomes revealed conserved regions not previously targeted by primers. two degenerate primers, flav f and flav r were designed from these regions and used to generate an base pair cdna product. the region amplified encoded part of the methyltransferase and most of the rna-dependent-rna-polymerase (ns ) coding sequence. one-step rt-pcr testing was successful using standard conditions with rna from over different flavivirus strains representing about species. the cdna from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template rna. conclusion: comprehensive testing has revealed the broad specificity of these primers. we briefly discuss the advantages and uses of these universal primers. most current molecular assays for flaviviruses use highly specific primers, which may only amplify from one species, or a range of closely related species [ ] [ ] [ ] [ ] . in a clinical or quarantine setting the presentation and potential exposures, including relevant travel history, are required to generate a differential diagnosis which is required before testing with specific primers. there is a real need to develop broad range pcr assays that can detect all flaviviruses. kuno [ ] reviewed this subject and compared several diagnostic protocols. his recommendation was a two stage process: initially utilizing broad range group-reac-tive primers to narrow the range of targets, followed by species-specific primers [ ] . many attempts to develop a systematic means for identifying flaviviruses have been made, including serology and non-serology based tests [ ] [ ] [ ] . due to the increased geographic distribution and severity of disease caused by members of the flavivirus genus, this need is becoming more pressing [ ] . the first report of a reverse transcriptase-pcr (rt-pcr) for the detection of multiple species was published in , with the use of species-specific probes targeting the nucleocapsid and envelope coding regions from four different dengue virus genomes [ ]. tanaka [ ] published the first universal primer pair specific for mosquito borne flaviviruses in ; the yf and yf primers targeted the ns / 'utr of the genome and were based upon the six flavivirus sequences available at the time. concurrently fulop [ ] designed a degenerate primer pair targeting conserved sites in the ns gene. these primers were successfully tested on thirteen different viruses including those in the tick-borne group and flaviviruses with no known vectors. pierre [ ] redesigned the yf and yf primer pair previously developed by tanaka, incorporating redundant bases to expand the range of viruses amplified. the primers emf and vd are unable to detect tick borne viruses because they lack the emf motif [ ] . in gaunt and gould designed a universal nested pcr, using six primers targeting the e gene, capable of amplifying cdna from flavivirus strains. the amplification of cdna was followed by restriction enzyme digestion to identify a range of virus species [ ] . the idea of designing primer sets relevant for diseases found in specific geographic regions has also been investigated by several groups. meiyu [ ] developed the djs and dja primer set targeting the ns gene; these were used in china to detect dengue virus (denv), and japanese encephalitis virus (jev). similarly the primers designed by tanaka (yf and yf [ ] were used to detect flaviviruses in brazil. however this primer pair failed to amplify bussuquara virus (bsqv), a virus native to brazil [ ] . flavivirus detection and taxonomy has recently become more difficult with the determination of the nucleotide sequence of tamana bat virus (tabv), and cell fusing agent virus (cfav) [ ] [ ] [ ] , and the discovery of kamiti river virus (krv). these viruses are currently classified as tentative members of the flavivirus genus [ ] , even though phylogenetic analysis indicates they are a distant sister group to the other recognised flaviviruses [ ] . they pose a problem for detection using pcr since primers depend on sequence conservation. gaunt and gould [ ] addressed this problem by using a nested pcr and increasing the degeneracy of primers, and demonstrated primers, with more than , different combinations in solution, were capable of detecting tabv. in the present study, we identified conserved sites and developed a universal, non-nested primer pair that amplifies cdna from each of the major subgroups of flaviviruses, and also tabv, under standard reaction conditions. the region of the ns gene amplified contained sufficient variability to allow differentiation of individual viruses. we discuss the advantages of this approach, over the known detection regimes for flaviviruses. no potentially useful conserved sites were identified in the first complete alignment, utilising all available sequences. however, the sequences of tabv, cfav and krv were identified as a divergent cluster, and once removed several conserved sites were found. the flav f and flav r primers were designed to complement sites in the ns gene that begin at residues and relative to the yfv genome (nc_ ). the conserved sites encoded amino acid sequences starting at residues and in the yfv polyprotein (np_ ), which do not correspond to any known conserved sites in flavivirus genomes. the primers have relatively low levels of degeneracy, with and different permutations respectively, discounting inosine positions, or with and permutations when inosines are counted as equivalent to four base degeneracy. to compensate for the primer multiplicity, a slightly higher primer concentration ( pmole per ul reaction) was used in the pcr. a cdna product approximately base pairs long was amplified from the rna of each of the viruses tested ( figure ). as expected there was variation in product size for some viruses, but products of the correct size were identified for every virus. the sizes estimated after gel electrophoresis corresponded closely with predicted size based on published sequences. when analysed by gel electrophoresis the cdna products displayed bands of varying intensities at ~ bp, although for some flaviviruses, products of multiple sizes were visible. each reaction contained µl of rna as template, thus the intensity of the product varied, presumably due to template concentration. all amplified products were sequenced and, on average, sequences from three reactions were used to traverse each cdna in both directions. full length sequence was obtained for viruses, and truncated sequence was obtained for denv ( bp), ugsv ( bp), bsqv ( bp), mvev ( bp), usuv ( bp), tyuv ( bp), tabv ( bp), yokv ( bp). cdna products of the expected size were obtained from aroav, bagv, bouv and lgtv although reliable sequence data was unavailable; thus these viruses have been excluded from this phylogenetic analysis. each product yielded sequence from a flavivirus ns gene as shown by blastn searches. flavivirus ns sequences occupied the top places in every blastn output. the majority of the sequences from the cdnas differed by to single nucleotide polymorphisms from the closest sequence with the same name in genbank. some viruses amplified had no relevant sequence data available on genbank, the identities of these viruses were further tested by phylogenetic analysis. a, b, c) representative pcr results showing the ~ bp fragment that was amplified the primers were tested on, and amplified cdna from, of the virus species listed in the mosquito-borne group, of the virus species in the tick-borne group and of viruses in the no known vector group [ ] . in total all of the species tested were amplified, seven flavivirus species have not been tested with these primers. cdna was also amplified from tabv, which was surprising as the available tabv sequences, and those of its closest relatives (cfav, krv), were removed from the alignments before the conserved sites were identified. the tabv sequences matched the flav f sequence at out of positions and none of the mismatches were located within the last bases of the ' end of the primer figure . the tabv sequences matched the flav r sequence at out of positions and mismatches were located at the ' end of the primer figure . despite this amplification involving mismatching with tamana bat virus rna, no cdna was amplified from the alphaviruses barmah forest virus, ross river virus or the nine respiratory viruses tested: influenza a virus, human coronavirus nl, human coronavirus oc , human ade-an alignment of the regions targeted by the flav f/flav r primers figure an alignment of the regions targeted by the flav f/flav r primers. identities are marked by a dot, gaps are marked by a dash, and nucleotide variants are shown. novirus, human bocavirus, human rhinovirus , or (data not shown). phylogenetic trees found using the sequences largely agreed with previously published trees [ , , ] in that the main subgroups were partitioned and the main known associations between species were found. sequences from the cdnas were paired with sequences recognized by the ictv or reference sequences from gen-bank ( figure ). the liv cdna sequence was the only exception, in that it appeared closer to the negv cdna sequence rather than the liv reference sequence; the liv reference sequence was the next closest sequence to the liv and negv cdna sequences. the relationship between liv and negv has previously been determined, thus it is unsurprising these viruses are more closely related to each other than to the reference sequence [ ] . all of the reference-cdna sister groupings in the trees were supported in all bootstrap resamples ( / ); some internal branches closer to the root were also well supported but others were poorly supported. the position of the slev sequence appeared to be anomalous, as it clustered with the members of the jev serogroup rather than rocv, as previously shown [ ] . phylogenetic analysis of the jev serogroup shows slev to be closely related to members of this serogroup [ , ] . recent phylogenetic studies using the e-ns -ns -ns sequence for rocv and other members of the jev serogroup shows slev to be closer to other members of the jev serogroup than the rocv [ , ] . the construction of phylogenetic trees based on shorter sequences, or different regions of the genome leads to different relationships between the viruses in particular the positions of slev relative the jev serogroup and rocv [ , , , ] we have described a novel primer set capable of amplifying bp from the ns genes from almost every recognised member of the genus flavivirus. since the amplified products represent % of the genome, this is sufficient sequence to determine the species of the virus and thus potentially to identify unrecognised flaviviruses. one major problem with degenerate primers is that the concentration of some permutations in the mixture is so small, due to their great multiplicity, that amplification is effectively inhibited. for any given viral rna target only a proportion of the primer may participate in the initiation of high efficiency extension in the early rounds of pcr. we believe that the redundancy of the flav f and flav r was insufficient to cause this problem [ ] . traditional serological methods based on neutralisation and fixed cell elisa have proven effective for identifying flaviviruses and indeed classifying them [ ] . however, some were not classified using this technology due to dif-a maximum likelihood tree of the cdna and references sequence found using an alignment of the base long region figure a maximum likelihood tree of the cdna and references sequence found using an alignment of the base long region. the mosquito-borne (m), tick-borne (t) and no known-vector (n) groups were partitioned as marked. ficulties in interpreting antigenic cross reactivity or failure to identify relatively close antigenic relationships that depend on epitopes encoded by regions of the genome that do not reflect the serological tests. moreover, serology is time consuming, requires highly experienced personnel and is less precise than nucleotide sequence determination. using molecular methods, it is now possible to analyse archival material and confirm the identification of tentatively identified flaviviruses. previous attempts to analyse the entire genus using pcr, have required multiple sets of primers. the capacity of the flav r and flav r primers potentially to amplify all flaviviruses makes them an invaluable diagnostic and taxonomic tool for virology. gaunt and gould, developed primers targeting the e gene [ ] . these primers did not amplify some species including, civ, crv, dbv, mmlv, ppbv and tabv [ ] . these viruses were all successfully amplified using the flav f/ flav r primers. primers targeting the ns gene have been developed and tested on a number of viruses including kunv, jev and yfv [ ] . bioinformatic analysis using sequence data available at the time, predicted that these primers would be unlikely to amplify products from tbev thus reducing their usefulness for a genome-wide study [ ] . the fu and cfd primers were tested on a large number of viruses; although six, covering the mosquito-borne kokv and sokv, tick borne (ksiv) and no known vector viruses rbv and svv, were unable to be reproducibly amplified using these primers. these viruses are highly divergent within the three major subgroups currently recognised in this genus [ , ] . the flav f/flav r primers amplified an bp product from each of these viruses. the ns gene has two distinct regions, a methyltransferase and a polymerase [ ] . we have targeted regions within two of the more highly conserved functional domains encoded by the flavivirus genome the primers designed in the present work have been widely tested, but there are six recognised viruses not included in the analysis; the bsl viruses, kyasanur forest disease virus and omsk hemorrhagic fever virus, the bsl viruses kedougou virus, san perlita virus and yaounde virus and the tentative members of the genus, cfav and krv. the primers amplified products from all tested flaviviruses. the ability of these primers to amplify previously 'unidentified' members of the flavivirus genus may demonstrate their capacity to define novel species. the protocol is robust and tolerates a range of template concentrations (greater than five orders of magnitude), primer concentrations, and pcr-cycle conditions (data not shown). the capacity of this reaction to amplify all fla-viviruses tested provides a potential tool capable of rapidly identifying endemic and exotic viruses, in a timely, cost effective manner, thus facilitating an appropriate response to epidemic outbreak, or surveys that may result in the discovery of new or novel flaviviruses. these primers also provide researchers with a tool to re-analyse archived samples that may no longer be infectious. in recent years viruses have been isolated from regions outside their known geographic distribution. jev was isolated in australia for the first time in . until this time the closest location to report human jev cases was bali. the outbreak of wnv in new york reinforces the importance of accurate and rapid diagnosis of exotic viral agents, as the virus was originally mis-diagnosed in serological tests. flaviviruses are emerging in new geographic regions as potential epidemic pathogens. thus, the importance of an accurate, rapid and reliable method for virus identification is becoming increasingly important. a major expansion of arbovirus surveillance and reporting systems has been implemented innorth america following the appearance of wnv. for example, arbonet reports surveillance data from humans, mosquitoes, birds, mammals and sentinel chicken flocks and the dataare integrated into a single reporting system [ ] . broad spectrum molecular tests such as that described in thispaper could make a significant contribution to such programmes. the changing global epidemiological environment is characterized by incursions of human populations into new environments, increasing overlap of the range of disease vectors with human habitation and concomitant exposure to a wider range of infectious agents [ ] . not only are humans changing land usage patterns and entering new disease environments [ ] , but rapid transportation of disease agents is constantly increasing between continents. outbreaks of emerging zoonoses, for example wnv in north america, and the threat of bio-terrorism with novel infectious agents, are no longer remote threats. the flav f and flav r primers have the potential to detect emerging, related flaviviruses without prior serological evidence or additional primer design. our approach should help reduce the confirmation time for viral infections. rapid detection at the genus level would enable informed policy measures to be implemented and this, in turn, may help disease management. primers were designed using a strategy similar to that used by vercruysse et al [ ] . all available full-length flavivirus sequences were retrieved from ncbi in march . sequences were sorted using bioedit [ , ] and aligned using clustalx [ ] . several divergent sequences were identified by examining neighbour joining trees found using clustalx and removed from the alignment. conserved regions were identified by calculating redundancy scores [ ] , and the average dominant base counts using an early version of the ncsf program and a window length of bases [ ]unpublished software, ; p. wayper and m.j. gibbs]. average dominant base counts were calculated by summing the number of occurrences of the most common base at each position in the window and averaging those counts across all positions in the window. the distance between conserved regions was taken into account when selecting conserved sites as was the potential for using mixed bases or deoxyinosines, to enhance bonding at variable positions [ ] . standard nucleotides were favoured close to the ' termini of the oligonucleotides. the primer sequences and their positions relative to the genome of yellow fever virus (nc_ ) are shown in table . primers were synthesised by geneworks (hindmarsh sa, australia). the primers target the end of the region encoding the methyltransferase and the start of the region encoding the rna-dependent rna-polymerase in the flavivirus ns gene. virus stocks produced in the molecular virology laboratory at the university of queensland, australia were prepared from the supernatant medium of infected ps-ek cell cultures (table ) . ps-ek cells were grown in dulbecco's modified eagle medium with % foetal bovine serum (gibco, carlsbad, california), u/ml penicillin and µg/ml streptomycin (invitrogen, carlsbad, california). to remove cellular debris, the supernatant medium was centrifuged at , rpm for min at °c. to increase the concentration, the virus particles were precipitated using a % polyethylene glycol (peg) nte solution ( . m nacl, mm tris-hcl, mm edta, ph . ). the virus-peg solution was stirred for - hours at °c, centrifuged at , rpm for hour at °c then resuspended in nte. viruses tested at oxford were prepared from the supernatant medium of infected % suckling mouse brain suspensions in pbs [ ] . viral rna was iso-lated from both sources using rnaqueous kit according to the manufacturer's protocol (ambion, austin, texas). one-step rt-pcr was performed using superscript iii in a µl volume (invitrogen, carlsbad, california) with touch-down cycling conditions [ ] . the final primer concentration in the rt-pcr was pmol per µl. a -min reverse transcription step was performed with incubations for minutes at each of °c, °c, °c and °c. enzyme activation at °c for minutes was followed by the touch down pcr. during cycling, denaturation and extension were performed at °c for seconds and °c for seconds respectively. annealing occurred for seconds during each cycle, with one cycle at each of the following temperatures of °c, °c, °c, °c, °c, °c, °c and °c. after the touch down stage, cycles with a °c annealing temperature, and then a final extension for minutes at °c completed the programme. the reaction was held at °c until processing then stored at - °c. the specificity of the primers was investigated by attempting amplification from cultures infected with viruses that are not flaviviruses, including barmah forest virus, ross river virus, influenza a virus, human coronavirus nl, human coronavirus oc , human adenovirus, human bocavirus, human rhinovirus , or and rna from virus free cell cultures. rt-pcr products were cloned into the pgem-t easy vector (promega, madison, wisconsin) according to the manufacturer's protocol. colonies were pcr screened for the presence of an insert. positive colonies were grown overnight in lb with µg ml - ampicillin. the plasmid was purified using a spin column kit (qiagen, eppendorf or invitrogen) according to the manufacturer's protocol. colony pcrs were performed using a step down protocol as described above although the extension temperature was °c (invitrogen, carlsbad, california). rt-pcr and pcr products were analysed on a % agarose gel containing ethidium bromide, and visualised using a uv transilluminator. purified plasmid was sequenced using abi bigdye terminator version . chemistry, on the ab xl sequencing platform. sp and t promoter primers were used for sequencing. each virus clone was sequenced twice or more in the forward and reverse directions. sequence data were assembled using contig express (invitrogen, carlsbad, california). sequences were then compared to the genbank non-redundant nucleotide database using blastn [ ] ; the programme identified the most closely matching sequences and produced alignments. species and strain names were matched between the genbank records and the virus isolates from which template rna was extracted. rt-pcr reactions were considered to have been successful if the highest scoring alignment was made with a sequence from the expected flavivirus and the correct region of the genome. publications were traced from the genbank files to confirm that the sequences had been correctly named. virus strain names were only used for those isolates where the strain had been identified by the international committee on taxonomy of viruses (ictv) [ ] . if there was no relevant sequence information available in the genbank database then the identification was based on phylogenetic analysis. sequences of known species and strains, identified by the ictv using their genbank accession codes, were compiled with the sequences from the amplified products; sequences were then aligned using the default single step progressive method of the program mafft version . [ , ] . maximum likelihood phylogenetic trees were found for the aligned sequences using the program phyml [ ] ; a general time reversible model was used, nucleotide frequencies and the proportion invariant nucleotides were estimated from the data, and variable rates were allowed at different positions with four rate categories. bootstrap analyses were done using the program paup version [ ] using the maximum parsimony and neighbour-joining methods. untranslated region; bsqv: bussuqara virus; cdna: complementary deoxyribonucleic acid; cfav: cell fusing agent virus; denv: dengue virus; dja: primer; djs: primer ; e gene: envelope protein encoding gene; elisa: enzyme linked immuno-sorbent assay ictv: international committee on taxonomy of viruses jev: japanese encephalitis virus; kokv: kokobera virus river virus; ksiv: karshi virus; kun: kunjin virus; l: ladder; ns : non-structural ; ntc: no template control paup: a phylogenetic programme; pbs: phosphate buffer saline; pcr: polymerase chain reaction; peg: poly-ehtylene glycol; phyml: phylogenetic programme; psek: porcine stable equine kidney cells; rbv: rio bravo virus ribonucleic acid; rt-pcr: reverse-transcription polymerase chain reaction; sokv: sokoluk virus; svv: sal vieja virus; tabv: tamana bat virus; tbev: tick-borne encephalitis virus; uv: ultra violet identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood rapid identification of flaviviruses based on conserved ns gene sequences rapid identification of flavivirus using the polymerase chain reaction identification of mosquitoborne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction universal diagnostic rt-pcr protocol for arboviruses an enzyme-linked immunosorbent assay for detection of flavivirus antibodies in chicken sera rapid subgroup identification of the flaviviruses using degenerate primer e-gene rt-pcr and site specific restriction enzyme analysis phylogeny of the genus flavivirus the west nile virus encephalitis outbreak in the united states detection of flaviviruses by reverse transcriptase-polymerase chain reaction with the universal primer set identification of brazilian flaviviruses by a simplified reverse transcriptionpolymerase chain reaction method using flavivirus universal primers the complete nucleotide sequence of cell fusing agent (cfa): homology between the nonstructural proteins encoded by cfa and the nonstructural proteins encoded by arthropod-borne flaviviruses genome sequence analysis of tamana bat virus and its relationship with the genus flavivirus isolation of a new flavivirus related to cell fusing agent virus (cfav) from field-collected flood-water aedes mosquitoes sampled from a dambo in central kenya rice cm: family flaviviridae genetic and phenotypic characterization of the newly described insect flavivirus, kamiti river virus molecular characterization of the japanese encephalitis serocomplex of the flavivirus genus brazilian flavivirus phylogeny based on ns nucleotide sequence of the envelope glycoprotein of negishi virus shows very close homology to louping ill virus complete genome characterization of rocio virus (flavivirus: flaviviridae), a brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in sao paulo state phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography highly degenerate, inosine-containing primers specifically amplify rare cdna using the polymerase chain reaction antigentic characterisation and classification of togaviridae use of ns consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses computer-assisted identification of a putative methyltransferase domain in ns protein of flaviviruses and lambda protein of reovirus linking human and animal health surveillance for emerging diseases in the united states: achievements and challenges arboviruses causing human disease in the australasian zoogeographic region the epizootics of kyasanur forest disease in wild monkeys during to rt-pcr using redundant primers to detect the three viruses associated with carrot motley dwarf disease bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools the gprime package: computer programs for identifying the best regions of aligned genes to target in nucleic acid hybridisation-based diagnostic tests, and their use with plant viruses base pairing involving deoxyinosine: implications for probe design evidence for the multiplication of scrapie agent in cell culture touchdown' pcr to circumvent spurious priming during gene amplification lipman dj: gapped blast and psi-blast: a new generation of protein database search programs mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood phylogenetic analysis using parsimony version genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy identification of new flaviviruses in the kokobera virus complex mjg and pjw were funded by the australian research council. slm was funded by the australian biosecurity crc, and uq gsrta. eag was funded by the eu fp research programme vizier. additional project funding was provided by biochip innovations pty. ltd. publication of this manuscript has been approved by the australian biosecurity crc. rtb is a director of biochip innovations pty ltd. key: cord- -wg qwmj authors: weiss, sabrina; dabrowski, piotr wojtek; kurth, andreas; leendertz, siv aina j.; leendertz, fabian h. title: a novel coltivirus-related virus isolated from free-tailed bats from côte d’ivoire is able to infect human cells in vitro date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: wg qwmj background: zoonotic transmission events play a major role in the emergence of novel diseases. while such events are virtually impossible to predict, wildlife screening for potential emerging pathogens can be a first step. driven by recent disease epidemics like severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebola, bats have gained special interest as reservoirs of emerging viruses. methods: as part of a bigger study investigating pathogens in african bats we screened animals for the presence of known and unknown viruses. results: we isolated and characterised a novel reovirus from blood of free-tailed bats (chaereophon aloysiisabaudiae) captured in in côte d’ivoire. the virus showed closest relationship with two human pathogenic viruses, colorado tick fever virus and eyach virus, and was able to infect various human cell lines in vitro. conclusion: the study shows the presence of a coltivirus-related virus in bats from sub-sahara africa. serological studies could help to assess its impact on humans or wildlife health. zoonotic transmission events play a major role in the emergence of novel diseases. while it is difficult to predict the emergence of zoonotic pathogens, wildlife screening of healthy animals for novel pathogens and evaluation of their capacity to induce disease can be a first step [ ] . among mammals, bats have gained special interest as potential reservoirs of emerging viruses such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebola [ , ] . a number of reoviruses (family reoviridae; respiratory, enteric, orphan virus) have been described in bats in europe and asia, all of which belong to the genus orthoreovirus in the spinareovirinae subfamily (e.g. [ - , , ] ). some of these have also been isolated from humans suffering from acute respiratory or gastrointestinal illness and likely originated from bats [ ] [ ] [ ] ] . as part of a bigger study [ , ] we investigated bats from sub-sahara africa for the presence of pathogens with zoonotic potential. the family reoviridae harbors viruses with a segmented double-stranded rna genome. it is currently divided into two subfamilies; the sedoreovirinae comprising six, and the spinareovirinae comprising nine genera [ ] . the genus coltivirus in the latter subfamily is comprised of two species only: colorado tick fever virus (ctfv) and eyach virus (eyav). ctfv is the etiologic agent of a febrile human disease, colorado tick fever, occurring in the rocky mountains in the western united states and canada [ ] [ ] [ ] . it is rarely fatal but can cause severe complications like encephalitis, haemorrhage, or pericarditis, especially in children [ ] . the related eyav was isolated in germany in and has been associated with human neurological disease by serological evidence [ , ] . animal reservoirs of coltiviruses are small mammals like rodents and lagomorphs and transmission to humans occurs by ticks of the family ixodidae [ , , ] . we describe a novel reovirus, designated taï forest reovirus (tfrv), which is closely related to coltiviruses, a genus not described in bats before, isolated from blood of african free-tailed bats (chaereophon aloysiisabaudiae). bats were captured with mist nets in november and december in the taï national park in côte d'ivoire. blood was taken from the wing vein and mixed with edta to prevent coagulation. animals were released immediately after the procedure. all samples were stored in liquid nitrogen, transported on dry ice and transferred to − °c at the robert koch-institut in berlin for long-time storage. primary virus isolation was achieved on veroe cells. for inoculation μl of blood from each of three individuals ch. aloysiisabaudiae was pooled. cells were grown to - % confluence in cm cell culture flasks. prior to inoculation cells were washed with phosphate buffered saline (pbs) followed by medium without supplements. cells were inoculated with blood diluted in pbs to a final volume of ml. after - h ml of medium including % fetal calf serum, % l-glutamine, % penicillin/streptomycin was added and cells were incubated at °c with % co . after h cells were washed with pbs and medium was replaced. cells were then incubated for days and monitored daily microscopically for the presence of a cytopathic effect (cpe). after days μl supernatant was used to infect fresh cells. a cpe was observed at day five in the second passage. to prepare virus stocks virus was grown in a cm flask and virus particles isolated by ultracentrifugation through a % sucrose solution for h at , rpm at °c in a beckmann ultracentrifuge (rotor sw ti, beckman coulter). for visualization of viral particles cells grown in a cm flask were fixed with glutaraldehyde (final concentration . %) over night at °c as soon as a cpe became visible. photographic documentation of samples was performed on a fei tecnai g transmission electron microscope (tem). viral rna was isolated using the qiaamp viral rna mini kit (qiagen) without carrier rna and initial sequence information was generated by particle-associated nucleic acid pcr [ ] and subsequent cloning and sanger sequencing of the amplicons. of the resulting sequences nine contigs and three individual reads showed homologies with two viruses, ctfv and eyav, when compared to genbank entries. the ctfv genome was used as reference genome to map contigs of the novel virus so as to get a possible arrangement, based on which sequence gaps were closed with primers directed outwards from known fragments (described in detail in [ ] ). segment ends of three identified segments (s , s , s ) were obtained by rapid amplification of cdna ends (thermo fisher). in parallel, rna was applied to next generation sequencing (roche), resulting in a total of , reads. reads were trimmed using trimmomatic version . [ ] and all trimmed reads shorter than bases were discarded. the resulting , reads were separated into pathogen and host reads using rambo-k version . [ ] (background reference: chlorocebus sabaeus, gcf_ . ; foreground references: eyach virus, nc_ -nc_ , and colorado tick fever virus, nc_ -nc_ ) using a k-mer length of and a cutoff score of − . , yielding , potential foreground reads. these reads were assembled together with the available sanger sequences using spades version . . [ ] . of the contigs reconstructed by spades, only were longer than bases. blastx of these contigs was performed against a custom database of the eyach virus and colorado tick fever virus genomes using geneious pro version [ ] , with contigs showing significant similarity to known segments. in an iterative process, all reads were mapped back to the contigs, and the mapping reads were re-assembled, until no new unambiguously mapping reads could be identified and contigs could not be extended anymore. finally, a last round of mapping all reads to all contigs was performed for error correction. to identify putative proteins encoded by the different segments possible open reading frames (orfs) were identified using the orf finder in geneious pro. putative gene functions were predicted based on homology to ctfv and the according uniprotkb entries [ ] . for phylogenetic inference sequences of rna dependent rna polymerase (rdrp) genes from representative viruses across different genera within the spinareoviridae were downloaded and aligned on protein level using the muscle algorithm [ ] as implemented in seaview version . . [ ] . to increase alignment quality conserved blocks were selected using the gblocks server [ ] , which resulted in an alignment of amino acids (aa). maximum likelihood (ml) tree was achieved using the phyml . server [ ] using the rtrev +g + f model as determined by smart model selection as implemented on the website. sequences of available putative rnamethyltransferase proteins from reoviruses were used for phylogenetic inference using the same pathway as described above. the resulting alignment consisted of aa and the model used for phylogenetic inference was lg + g + f. assembled sequences of the novel virus showed homology to two viruses exclusively, ctfv and eyav, which together form the genus coltivirus. this was confirmed by tem pictures showing particles of approximately nm in diameter and typical inner and outer icosahedral capsids, which are characteristic for reoviruses (fig ) . coltiviruses have double stranded rna genomes comprised of genomic segments with sizes ranging from to nucleotides (nt). using sanger and sequencing methodology nine segments of the novel virus could be assembled. segments , , and were completely assembled whereas for segments , , , , and ends are missing. for segment two long contigs were assembled but not combined to one long contig. no sequence information was identified for segments , and . table provides comparison between identified segments those of ctfv and eyav. table provides an overview about similarities between the segments and ctfv and eyav segments. the novel virus was tentatively named taï forest reovirus (tfrv), based on the origin of its host. with few exceptions reovirus segments commonly contain one orf coding for a single protein, flanked by noncoding regions and conserved terminal sequence motifs. identified segments of tfrv are monocistronic with the exception of one segment, which putatively contains two overlapping orfs, mediated through an opal stop codon, followed by a cytosine residue. this construction allows for two proteins of different lengths, but beginning with the same start codon, a shorter viral protein (vp ) and a longer vp ' protein [ , , ] . segment lengths and putative start codons in comparison to ctfv and eyav are listed in table virus protein (vp) of tfrv contains rna polymerase domains (uniprot q dsq ) and putatively encodes for a rna dependent rna polymerase. this is supported by the presence of two functional motifs that are present in all reovirus rdrp genes [ ] : motif sg (positions - for ctfv and eyav, positions - for tfrv) and motif gdd (positions - for ctfv and eyav, - for tfrv). vp on segment might code for a rna methyltransferase according to uniprot (q enl ), vp on segment for a membrane protein (uniprot q enl ), the orfs vp and vp ' on s for a structural and nonstructural protein (uniprot o and o ), respectively, and vp for a microtubule-associated protein (uniprot q enk ). based on sequence information of segment , a quantitative real-time pcr (qpcr) assay was designed to determine the viral load in the original samples and to screen all bat blood samples available from taï national park. in total, samples from bat genera were tested. tfrv was only detected in samples from three chaerephon bats the rdrp gene encoded on segment and the putative rna-methyltransferase genes encoded on segment were used for phylogenetic analysis, which confirm the relationship between tfrv and coltiviruses (fig ) . to estimate the possible host range of tfrv it was used for inoculation of various cell lines. the virus was able to induce a cpe on c / insect cells and on various mammalian cell lines (fig ) : primate kidney cells (veroe ), a fruit bat cell line originating from rousettus aegyptiacus (r t), and two human cell lines, lung fibroblasts (mrc- ) and liver cells (hep ). we have isolated a novel reovirus from blood of freetailed bats from côte d'ivoire that clusters together with coltiviruses in phylogenetic analysis. the isolation of tfrv from blood is in line with the observed relationship to coltiviruses. ctfv, the type species of coltiviruses, replicates in erythropoietic cells and produced virus is maintained in red blood cells [ ] . the level of aa identity between homologous proteins of different strains within various genera across the reoviridae family is between %- % [ ] . % aa identity of the rdrp with both, ctfv and eyav, suggesting that tfrv belongs to the same genus, namely coltivirus. for those segments identified, comparisons between tfrv and coltiviruses show identities ranging from % to %. between homologous segments of ctfv and eyav identities vary between % and %, exceptions being segments and , where identities are % and % only. assuming similarities between tfrv and ctfv or eyav are even lower, this might explain difficulties to identify, or accurately annotate, contigs belonging to segments and . coltiviruses are known to have a large host range in vivo, infecting ticks as well as a variety of mammals [ ] , so the host range observed in vitro for tfrv is not surprising. however, the induction of a cpe on a wide range of mammalian cells, including human cells, together with its close phylogenetic relationship to human pathogenic viruses, allows for speculation about the ability of tfrv to infect humans. febrile symptoms, as caused by ctfv, are common in tropical regions and can account for a number of different diseases. in africa, based on present data we cannot infer whether ch. aloysiisabaudiae is an accidental host or represents the true reservoir of tfrv. no tfrv was detected in any other bat species, including one individual of the related species ch. russata. ch. aloysiisabaudiae is widely distributed across the sub-saharan africa. besides the forest zones in côte d'ivoire and western ghana, it is also present in central africa along a stretch from cameroon to south sudan and uganda. since populations are spatially separated it would be of interest to look for tfrv in central african bats to further explore its epidemiology. the screening of ticks could also be of interest. ixodid ticks are the vector for infecting humans with ctfv and eyav and are also present in africa [ ] . the close relationship of the viruses and the observation that tfrv infects insect cell lines in vitro allows for the possibility that it also requires ticks or another vector to spread. despite the fact that this would limit the possibility for transmission to a region with a geographical overlap of bats, vector, and humans, the presence of the virus in blood raises concern about transmission by bloodfeeding vectors. tfrv is able to infect cells from various mammals in vitro and it might be that other mammals or vectors also play a role in the virus' ecology. development of a serological test to screen animals and humans from the taï region for antibodies against tfrv would help to shed light on the potential transmission of this novel reovirus in the taï forest region and along the distribution of ch. aloysiisabaudiae. we described a putative novel member of the coltivirus family. to our knowledge, tfrv represents the first coltivirus isolated from bats and on the african continent. with respect to the pathogenic potential of coltiviruses further screening of wildlife and possible vectors could help to learn about the epidemiology of the virus and to assess its potential impact on human and animal health. targeting surveillance for zoonotic virus discovery bats and their virome: an important source of emerging viruses capable of infecting humans assessing the evidence supporting fruit bats as the primary reservoirs for ebola viruses a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient isolation and characterization of three mammalian orthoreoviruses from european bats a novel reovirus isolated from a patient with acute respiratory disease isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in china identification of mammalian orthoreovirus type in italian bats xi river virus, a new bat reovirus isolated in southern china high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in europe hantavirus in bat henipavirus-related sequences in fruit bat bushmeat, republic of congo virus taxonomy: classification and nomenclature of viruses the etiology of colorado tick fever sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus coltivirus (family reoviridae) de lamballerie x: genus coltivirus (family reoviridae): genomic and morphologic characterization of old world and new world viruses in medical microbiology eyach -an arthorpodborne virus related to colorado tick fever virus in the federal republic of germany antibodies against some arboviruses in persons with various neuropathies isolation of colorado tick fever virus from rodents in colorado ecology of colorado tick fever characterization of virus isolates by particle-associated nucleic acid pcr identification and characterisation of emerging viruses in freeranging bats from sub-saharan africa trimmomatic: a flexible trimmer for illumina sequence data rambo-k: rapid and sensitive removal of background sequences from next generation sequencing data spades: a new genome assembly algorithm and its applications to single-cell sequencing geneious basic : an integrated and extendable desktop software platform for the organization and analysis of sequence data reorganizing the protein space at the universal protein resource (uniprot) muscle: multiple sequence alignment with high accuracy and high throughput seaview version : a multiplatform graphical user interface for sequence alignment and phylogenetic tree building improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . termination and read-through proteins encoded by genome segment of colorado tick fever virus isolation of eyach virus (reoviridae, colorado tick fever group) from ixodes ricinus and i. ventalloi ticks in france we thank the authorities in côte d'ivoire for their long-term support, especially the ministry of the environment and forests as well as the ministry of research, the directorship of the taï national park, and the centre suisse de recherches scientifiques en côte d'ivoire in abidjan. for committed support during fieldwork we thank stefan petterson and the field assistants of taï national park. stephan becker, markus eickmann and verena krähling we would like to thank for support during initial laboratory work, the sequencing laboratory at rki and kevin merkel for technical assistance. no specific funding was obtained for this study.availability of data and materials sequences from this study are available on genbank under the following accession numbers: authors' contributions sw and fhl planned the study. sw conducted the experiments and wrote the paper. pwd contributed to data analysis, specifically data. ak performed microscopy and provided advice during the study. sjl conducted fieldwork and contributed to writing the paper. fhl conducted fieldwork and provided overall project management. all authors read and approved the manuscript. no ethical approval was needed to conduct this study. the study was conducted in compliance with national and international guidelines. not applicable. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -ba dus j authors: he, lei; zhang, yan-ming; dong, ling-juan; cheng, min; wang, jing; tang, qing-hai; wang, gang title: in vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin rnas targeting the orf gene date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ba dus j background: transmissible gastroenteritis (tge) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. currently, the vaccines for it are only partially effective and no specific drug is available for treatment of tge virus (tgev) infection. rna interference has been confirmed as a new approach for controlling viral infections. in this study, the inhibitory effect of short hairpin rnas (shrnas) targeting the orf gene of tgev on virus replication was examined. results: four theoretically effective sequences of tgev orf gene were designed and selected for construction of shrna expression plasmids. in the reporter assays, three of four shrna expression plasmids were able to inhibit significantly the expression of orf gene and replication of tgev, as shown by real-time quantitative rt-pcr analysis of viral orf and n genes and detection of virus titers (tcid( )/ml). stable swine testicular (st) cells expressing the shrnas were established. observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shrnas were capable of protecting st cells against tgev destruction, with high specificity and efficiency. conclusions: our results indicated that plasmid-transcribed shrnas targeting the orf gene in the tgev genome effectively inhibited expression of the viral target gene and viral replication in vitro. these findings provide evidence that the shrnas have potential therapeutic application for treatment of tge. transmissible gastroenteritis (tge) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. these syndromes are especially serious in neonatal piglets aged < weeks old, frequently leading to a mortality as high as %; even surviving older pigs generally show growth retardation and low reward to feeding [ ] [ ] [ ] . tge causes enormous economic loss annually worldwide. currently, several vaccines for prevention of tge are available, but their efficacy is variable. both the attenuated and inactivated vaccines are partially effective; attenuated tge virus (tgev) vaccines have the risk of reverting to a virulent form and may even induce an adverse reaction, and the inactivated ones are poorly protective in swine [ , ] . moreover, newborn piglets infected with tgev may die within - days, whereas the vaccines for tgev do not provide effective protection at days after administration [ ] . however, no cure for tge is presently available apart from symptomatic treatment. rna interference (rnai) has been confirmed as a posttranscriptional gene silencing mechanism, which is widely considered to be the major antiviral system in plants and insects [ , ] . the effective inhibition of replication of several rna and dna viruses in animal cells also has been reported by means of rnai [ ] [ ] [ ] [ ] [ ] . therefore, rnai may be developed as a potential therapy for tge. tgev is a member of the genus alphacoronavirus in the family coronaviridae. it is a positive-sense, ssrna virus with a . -kb genome that contains a leader sequence at the end and a poly(a) tail at the end, and encodes four structural proteins [spike (s), envelope (e), membrane (m) and nucleoprotein (n)] and five nonstructural proteins (replicase a, b, a, b and protein ) [ ] [ ] [ ] . the genome itself, together with six sub-genomic mrnas transcribed discontinuously, forms a nested set of rnas of different lengths with co-terminal ends [ ] . orf is located at the end of the genome. it encodes a -amino-acid, . -kda hydrophobic protein, and has been reported to play a role in the process of membrane-associated replication complexes and/or virion assembly [ , ] . recently, more studies have shown that the deletion of tgev orf by reverse genetics may be related to the viral virulent and promotes an intensified dsrna-activated host antiviral response [ ] . compared to other viral proteins' gene (such as proteins s, e, m or n), the orf region is relatively conservative and rnai targeting to orf gene will result in the degradation of both the sub-genomic mrna for orf and the other sub-genomic mrnas [ , ] . besides, data showed that the ' end of tgev genome (orf gene and the downstream gene) could interact with host cell proteins and played a positive role in the replication of tgev [ , ] . according to the characteristics of rnai, the downstream gene will be degraded with the orf gene. then the interaction of ' end of tgev genome and host cell proteins will be interrupted and the viral replication will be reduced. thus, it is helpful to use rnai against it as a new therapeutic option. here, we demonstrate that rnai targeting of the orf gene of tgev, introduced by short hairpin rnas (shrnas), is capable of inhibiting virus replication and protecting swine testicular (st) cells from the destruction induced by tgev, which may be not only a new anti-tgev strategy, but also a new approach to the study of its pathogenesis. relative quantifications of both the orf and n genes were performed by real-time quantitative rt-pcr. comparative threshold (ct) cycle values in three independent experiments were calculated by the ct method, and the average relative amount of orf gene in each sample is represented in figure . the relative amount of orf gene in mock control cells was regarded as . , where the relative amounts of orf gene in cells infected with tgev after being transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were . , . , . , . and . , respectively, and in cells infected with tgev before transfection was . , . , . , . and . , respectively. the sequence-specific shrna pgpu -gfp/ reduced the amount of orf gene by approximately % and %, which was better than with the other three plasmids. real-time quantitative rt-pcr of the n gene ( figure ) for cells infected with tgev after transfection with those shrnas was . , . , . , . and . , respectively. this indicated that these sequence-specific shrnas had approximately %, %, %, % and % reductions in tgev genome (included sub-genomic mrnas, except the shortest one). although the levels of viral genome in the cells infected with tgev before being transfected with shrnas were . , . , . , . and . , corresponding to a %, %, %, % and % reductions in tgev rna. taken together, rnai against orf gene showed a dramatic reduction in the tgev viral. the % tissue culture infective dose (tcid ) assay was performed to examine the effect of sirna on production of viable virus. figure shows that the titers of tgev were . , . , . , . and . tcid /ml in cells infected with tgev after being transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc at h post-infection, respectively, and the titers of tgev in cells infected with tgev before being transfected with shrnas were . , . , . , . and . tcid /ml. these data indicated that rnai against orf gene reduced the progeny virus production significantly; pgpu -gfp/ showed a maximum inhibition, whereas pgpu -gfp/ and pgpu -gfp/ showed partial virus replication inhibition, with pgpu -gfp/ being the least effective shrna. based on the results obtained by real-time quantitative rt-pcr and determination of tcid , cells stably expressing pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were selected by using g (pgpu -gfp/ abandoned). green fluorescence protein (gfp) was used as a reporter and cells were screened until % of them showed green fluorescence. as shown in figures and , a similar virus replication inhibition trend was observed in the cell lines in comparison with the transiently transfected cells. the three shrna-expressing cells exhibited potent ability in silencing tgev rnas, whereas cells expressing pgpu -gfp/nc showed a slightly nonspecific effect. to study the protective effect of shrnas against tgev destruction, the mts cell proliferation assay was performed on st cells stably expressing shrna plasmids. as the absorbance is proportional to the number of living cells in culture, it was measured at nm by using an elisa reader at h post-infection (including h incubation with mts). mean od of solutions in st cells stably expressing pgpu -gfp/nc and in control st cells was . and . , respectively, whereas in st cells expressing pgpu -gfp/ , pgpu -gfp/ or pgpu -gfp/ were . , . and . , respectively ( figure ). this showed that st cells expressing shrnas, especially pgpu -gfp/ , were protected from tgev destruction, leading to a significant increase in living cells compared with cells stably expressing pgpu -gfp/nc, or st control cells. to further investigate the effect of shrnas on protecting st cells against tgev-induced destruction, the cytopathic effect (cpe) and apoptosis of cells stably expressing shrnas were examined by fluorescence microscopy. the apoptotic cells were characterized by condensed nuclei, and were colored blue by fluorescent dye hoechst , whereas the cpe in cells that changed from cell fusion to lysis, and thereby formed large bodies, were stained red by propidium iodide (pi). as shown in figure , the negative control pgpu -gfp/nc had no apparent inhibitory effect on tgev-induced cpe and apoptosis, because many cells formed large bodies and showed condensed nuclei. in contrast, cells expressing pgpu -gfp/ had few condensed nuclei, and almost all cells expressing pgpu -gfp/ were capable of maintaining cpe resistance, because there were no apparent pi-stained cells. however, a small area of mild cpe was seen in the cells expressing pgpu -gfp/ and pgpu -gfp/ , indicating that these plasmids were less effective than pgpu -gfp/ at protecting st cells against tgev-induced destruction. although several veterinary coronavirus vaccines are currently available, their efficacy is variable. among them, the infectious bronchitis virus vaccine is very effective for chickens [ ] , whereas the canine and porcine vaccines are only partially effective. furthermore, there is currently no effective antiviral treatment against these virus infections [ ] ; therefore, rapid and potent anti-tgev therapeutic agents are urgently needed. rnai technology provides an important methodology for rational drug design and gene therapy for many viral diseases, which has proven to be a potent tool for host protection against viral infection, suppression of viral genome transcription, and blocking viral replication [ , ] . rnai can be introduced into the cells using two different approaches. the first is chemically synthesized sirnas. cells transfected with chemically synthesized sirnas can achieve rapid and effective silencing of a target gene, but the effects are transient. second, shrnas, which can be cleaved by dicer to produce sirnas in the host cell, can circumvent the disadvantages of chemically synthesized sirnas by using stably transfected plasmids or virus vectors [ ] [ ] [ ] . the present study demonstrated the use of rnai against tgev via shrna-expressing plasmid vector pgpu -gfp, which significantly reduced viral genomic rna replication and protected st cells from tgev destruction, by targeting orf gene. rnai is highly sequence-specific and requires % identity between the target and targeting sequences to achieve virus clearance from cell culture [ ] [ ] [ ] . the application of shrnas targeting the conserved region of the gene is one way to overcome the lack of knowledge of the target sequence. to ensure that shrnas can be used for a wide range of virus strains, we evaluated the cross-inhibitory capabilities of these shrnas by conducting multiple alignments of tgev orf gene sequences, based on the available sequences in genbank. our results showed that pgpu -gfp/ , pgpu -gfp/ and and pgpu -gfp/ could cover % ( / ), % ( / ) and % ( / ) of tgev strains, respectively (data not shown). interestingly, we have also conducted multiple alignments of the sequences among tgev, feline coronavirus virus and canine coronavirus, and have shown that they are also conservative. this conservative characteristic of the sequences between the three viruses means that they have greater potential application for the treatment of the diseases caused by these viruses, which also indicates that further studies are necessary to search for the cross-inhibitory effects in a range of tgev strains and other coronaviruses. in our study, two strategies were used to detect the inhibitory effect of shrnas on both the orf gene and tgev genome. the first approach was that cells were infected with tgev after being transfected with shrnas, in which the shrnas were used as preventive substances. as shown in figures and , there was a marked decrease in both the orf and n genes, which could be amplified simultaneously from all the same regions within the tgev genome rna and sub-genomic mrnas (except the shortest one). the reduction showed that the viral genome rna and its transcripts were decreased significantly. a similar suppression pattern was observed for virus titers. in the second strategy, shrnas of orf gene were used as therapeutic agents and transfected after the cells were infected with tgev. almost all the cells maintained their normal morphology before they were collected for real-time rt-pcr. the orf and n genes were reduced significantly when the shrnas were transfected at h after tgev infection (figures and ) . more experiments were carried out at and h after tgev infection, but the results were not as remarkable as those after h (data not shown), which indicates that shrnas affect the initial stage of tgev infection. in the study by ortego et al., recombinant tgev with orf gene deletion replicated in cell culture with similar efficiency to the wild-type virus, and stably maintained the modifications introduced into the genome [ ] . this observation seems to contrast with the results of the present study, which provides evidence that rnai of the orf gene could lead to decreased virus replication. there are three possible reasons for this. first, the genome of parental tgev could be degraded directly because it consists of a positive-sense ssrna. second, all the other sub-genomic mrnas could be degraded as the orf gene is included in all the sub-genomic mrnas. as shown in figures and , n gene was significant decreased in the shrna expressing cells. besides, the end of the tgev genome gene, the downstream gene of orf gene, could be degraded with the orf gene according to the characteristics of rnai, and its degradation could delay the viral replication, because it has been reported that it could interact with host cell proteins and have a great influence on the replication of tgev [ ] . in the study by cruz et al., it was reported that tgev without orf enhanced virulence in infected piglets [ ] . we speculate that this phenomenon won't happen on the rnai treated pigs, because the rnai method is different from the reverse genetics technique. by the rnai used in this study, the other viral sub-genomic mrnas were degraded (figure and ) . while the reverse genetics deletion will only affect the orf gene itself. nevertheless, whether the technology could be used in vivo still needs more explored. to elucidate the protection of shrnas against tgev in more detail, st cells stably expressing the shrnas were established. tgev replication in these cells was observed through detection of virus titers and real-time quantitative rt-pcr. cells expressing pgpu -gfp/ showed % inhibition in the viral genome, whereas pgpu -gfp/ showed up to % and pgpu -gfp/ % inhibition (figure ) . tgev destroys st cells in two ways, by apoptosis and necrosis [ , ] . therefore, cpe and apoptosis were analyzed. examination of tgev-induced cpe indicated that st cells were very sensitive to tgev infection. cells expressing pgpu -gfp/nc formed large bodies and were stained red by pi (figure ) , whereas cells expressing pgpu -gfp/ were not stained red, indicating that the cell membrane was intact. compared with the condensed, irregular nuclei of pgpu -gfp/nc-expressing cells, cells expressing pgpu -gfp/ were completely protected from apoptosis and the nuclei showed normal morphology. the sequence-specific shrnas, especially pgpu -gfp/ , exhibited potent ability to protect st cells from tgev-induced destruction. our present results indicate a close relationship between inhibition of tgev replication and cell viability by shrnas. our data also suggest that sirna targeting orf gene can elicit viral rna from infected cells and potentially offers an efficient therapeutic option for tgev infection. taken together, our results indicate that plasmidtranscribed shrnas targeting the orf gene in the tgev genome can inhibit expression of viral target genes and viral replication in st cells. this method merits further investigation in animal studies to define its therapeutic potential, and whether the technology could be used in vivo for anti-tgev therapy is still under investigation. st cells were cultured in high-glucose dulbecco's modified eagle's medium (dmem; gibco, uk) containing % heat-inactivated fetal calf serum (hyclone, china) and antibiotics ( μg/ml streptomycin and u/ml penicillin); the culture medium was replaced every days. tgev h strain was obtained from the national control institute of veterinary bioproducts and pharmaceuticals (beijing, china) and propagated in st cells. sequences from the orf gene of tgev h strain (genbank accession no: fj ) were designed based on the website sirna designing tools (http://www. ambion.com/techlib/misc/sirna_finder.html and https:// rnaidesigner.invitrogen.com/rnaiexpress/). the sequences were analyzed by blast to ensure that they did not have significant nucleotide sequence homology with the swine genome, but shared % identity with the other published sequences of tgev strains, from which four theoretically effective sequences at nucleotide positions - (orf - ), - (orf - ), - (orf - ) and - (orf - ) were selected. a nonspecific sequence (nc) was also scanned by blast analysis and served as a negative control. these five sequences are listed in table . all the sequences were arranged in the following alignment: bbsІ + sense + loop + antisense + termination signal + bamhІ and cloned into the pgpu -gfp vector to make shrna expressing plasmids: pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc. twenty-four hours before being transfected, st cells were seeded into six-well dishes in high-glucose dmem + % fetal bovine serum (fbs) without antibiotics. when they reached - % confluence, cells were transfected with μg/well pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc by lipofectamine (invitrogen, carlsbad, ca, usa) according to the manufacturer's recommendations. after being incubated at °c for h, the transfection complex was removed and the medium was replaced by high-glucose dmem with % fbs. the cells were inoculated with tgev at tcid h later. the plasmids contained the gfp gene sequence. therefore, gfp was expressed allowing a good discrimination between transfected and non-transfected cells. the transfection efficiency was determined by monitoring the percentage of gfp-expressing cells within the live cell population, and it showed that nearly % of the transfected cells were positive. cpe was evaluated and the cell images were captured under an inverted fluorescence/phase-contrast microscopy (nikon, japan) at different time points post-infection. the cell cultures were collected for a real-time quantitative rt-pcr analysis at h post-infection. to assess the influence of shrnas on tgev replication, the n gene of tgev was used as a standard for the tgev genome. another pair of primers was synthesized for quantification of the tgev genome in real-time rt-pcr: forward, -ggaagatggcgaccagatag- and reverse, -ccacttctgatggacgagca- . total rna of culture supernatants was isolated as described above, and a real-time quantitative rt-pcr was conducted using a sybr exscript™ rt-pcr kit (takara bio), according to the manufacturer's instructions. real-time rt-pcr was performed and the procedure was similar to that described above, expect that the reaction temperature for annealing was °c. data were analyzed according to the ct method, and the tests were performed in triplicate. tgev cultures in sirna-transfected cells were collected h after viral infection. after three freeze-thaw cycles, the cultures were serially diluted -fold from - to - , and added to st cells at - % confluence in -well plates. each dilution was added to four wells. after days of infection, the tcid was calculated by the reed-muench method. the pgpu -gfp vector carries the neomycin resistance gene; therefore, st cells stably expressing shrnas were selected using g . gfp in the plasmids was used as a reporter during the selection efficiency analysis. the st cells were seeded into six-well plates h before being transfected (up to - % confluence). cells were transfected with pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc by lipofectamine as described before, and propagated in selection medium containing μg/ml g until % of the surviving cells stably expressed gfp. st cells stably expressing pgpu -gfp/ , pgpu -gfp/ , pgpu -gfp/ and pgpu -gfp/nc were seeded in -well plates at - % confluence and challenged with tgev at tcid . cell viability was assessed by adding μl/well of mts (promega, usa) to cell cultures, according to the manufacturer's instructions, at h after viral infection. after incubation with mts for h, light absorbance of each well was measured at nm. each measurement was performed six times, and the experiment was repeated three times. st cells stably expressing shrnas were seeded in sixwell tissue culture plates at - % confluence and challenged with tgev at tcid . thirty hours after infection, the cells were washed with hank's balanced salt solution (hbss) and incubated with hoechst ( ng/ml) at °c for min, and then washed three times with hbss. cell were then incubated with pi ( ng/ml) at °c for min and washed with dmem without serum. images were viewed by fluorescence microscopy (nikon, japan). transmissible gastroenteritis of pigs genetic basis for the pathogenesis of transmissible gastroenteritis virus binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins mucosal immunisation and adjuvants: a brief overview of recent advances and challenges increased litter survival rates, reduced 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replication and virulence protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/ safety and efficacy of a modified-live canine coronavirus vaccine in dogs simultaneously inhibition of hiv and hbv replication through a dual small interfering rna expression system in vitro inhibition of csfv replication by retroviral vector-mediated rna interference a transgenic approach for rna interferencebased genetic screening in mice a system for stable expression of short interfering rnas in mammalian cells a large-scale rnai screen in human cells identifies new components of the p pathway short interfering rna confers intracellular antiviral immunity in human cells targeting marek's disease virus by rna interference delivered from a herpesvirus vaccine clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas transmissible gastroenteritis virus induces apoptosis in swine testicular cell lines but not in intestinal enterocytes the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and − during tgev-induced apoptosis the authors declare that they have no competing interests. lei he took part in all the experiments, and wrote the manuscript. yan-ming zhang designed all the experiments. ling-juan dong and min cheng participated in plasmid construction, cell transfection and confocal microscopy. jing wang made a major contribution to the mts assay. gang wang and qing-hai tang carried out the cell culture and real-time rt-pcr. all authors read and approved the final manuscript. key: cord- -vtjsfi c authors: sultankulova, kulyaisan t.; kozhabergenov, nurlan s.; strochkov, vitaliy m.; burashev, yerbol d.; shorayeva, kamshat a.; chervyakova, olga v.; rametov, nurkuisa m.; sandybayev, nurlan t.; sansyzbay, abylay r.; orynbayev, mukhit b. title: new oligonucleotide microarray for rapid diagnosis of avian viral diseases date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: vtjsfi c background: we developed a new oligonucleotide microarray comprising identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (aiv), newcastle disease virus (ndv), infection bronchitis virus (ibv), and infectious bursal disease virus (ibdv) in single- and mixed-virus infections. the objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. methods and results: the paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cdna on the microarray and its specificity tested with use of aiv, ndv, ibv, ibdv strains as well as biomaterials from poultry. sensitivity and specificity of the developed microarray was evaluated with use of specimens of biological material: cloacal swabs from sick birds and tissue specimens from dead wild and domestic birds, as well as with use of aiv, ndv, ibv and ibdv strains, different in their origin, epidemiological and biological characteristics (ribsp microbial collection). this microarray demonstrates high diagnostic sensitivity ( . % within % ci limits . – %) and specificity ( %). specificity of the developed technique was confirmed by direct sequencing of np and m (aiv), vp (ibdv), s (ibv), np (ndv) gene fragments. conclusion: diagnostic effectiveness of the developed dna microarray is . % and therefore it can be used in mass survey for specific detection of aiv, ndv, ibv and ibdv circulating in the region in the course of epidemiological surveillance. rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic methods is proposed. intensive poultry farming leads to higher risk of infectious disease emergence causing great economical losses. boundary spanning between clinical manifestations of different agents is peculiar to the course of many infections nowadays. more and more infectious diseases progress in association with different microorganisms and it effects significantly the clinical manifestation and differential diagnosis of the disease. currently, the viral infections such as avian influenza, newcastle disease, infectious bronchitis, and infectious bursal disease, etc., are a potential threat to poultry farming in the republic of kazakhstan. monitoring these economically significant avian diseases is the question of the day for poultry industry. avian influenza virus belongs to the orthomyxoviridae family, influenza a virus genus. from the beginning of year the disease outbreaks were recorded in countries [ ] . different aiv strains can cause to % mortality among poultry. the agent of the newcastle disease is an rnacontaining virus, a member of the paramyxoviridae family, rubulavirus genus. in countries reported newcastle disease cases to the oie [ ] . in poultry industrial farms, all infected birds need to be sacrificed due to threat of dissemination of the infection across countries [ ] . the agent of the infectious bursal disease is rnacontaining virus of avibirnavirus genus in birnaviridae family. in outbreaks of the infectious bursal disease practically the entire population is affected and the lethality rate can approach % [ ] , the reconvalescent birds become susceptible to the majority of infectious diseases of viral and bacterial etiology [ ] . the causative agent of infectious bronchitis is an rna-containing coronavirus avia of coronavirus genus in coronaviridae family [ ] . economical losses due to infectious bronchitis is composed of reduced egg and meat productivity, compulsory slaughter of sick birds, high death rate in young population. when the infection circulates in the farm for the first time the lethality rate can reach % [ ] . currently, standard immunological methods [ ] or methods based on polymerase chain reaction (pcr) [ , ] are widely used to identify the above mentioned viruses. unfortunately, they can detect only one agent in a specimen. there are also multiplex rt-pcr assays that make possible simultaneous detection of more than one infectious agent by using multiple primer pairs. advantage of the multiplex rt-pcr is in combination of sensitivity and quickness of pcr alongside with elimination of need to test clinical specimens for each agent separately [ , ] . avian viruses can cause diseases independently, in alliance with each other or in association with bacterial agents [ ] . thereby, rapid and sensitive methods of detection are required that are able to differentiate viral infections for surveillance of newly emerging avian viruses as well as for disease control. application of dna microarray technology that makes possible multivariate analysis of genetic material is a highly promising way for simultaneous detection of several agents (aiv, ndv, ibv and ibdv) in one specimen. the paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescentlabelled viral cdna on the microarray and its specificity tested with use of aiv, ndv, ibv, ibdv strains as well as biomaterials from poultry. the objective of this study is to develop an oligonucleotide microarray for rapid diagnosis of avian influenza, newcastle disease, infectious bronchitis, and infectious bursal disease that will be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. four aiv and ndv strains, ibv and ibdv strains from the ribsp (me&s rk/sc) microbial collection were used in the study (see table for the list of these strains). one-hundred and twenty-two samples ( cloacal swabs from sick birds and tissue samples from dead ones) were delivered by veterinarians from different regions of kazakhstan to rge ribsp in the routine epidemiological surveillance for diagnosing sickness and death of birds (table ) . rna was extracted from virus-containing material with trizol ("invitrogen", usa) according to the manufacturer's instruction. for selection of oligonucleotide primers and probes as microarray components the representative sample from international data base of ncbi (national center for biotechnological information) genbank (https:// www.ncbi.nlm.nih.gov/) containing genomes of aiv, ndv, ibv and ibdv was used. all full-sized encoding sequences of virus nucleotides were aligned by using clustal w algorithm in mega . software by method of progressive multiplex alignment. oligonucleotide probes with optimal physicochemical characteristics were selected with use of oligowiz . and picky . programs. specificity of the chosen oligonucleotide primers and probes was tested with use of blast (basic local alignment search tool) program that could compare the existing sequence with sequences in database in ncbi blast-analysis (ncbi, https://blast.ncbi.nlm.nih.gov/ blast.cgi). oligonucleotide primers and probes were synthesized in dna/rna synthesizer h- (k&a laborgeraete, germany) according to the manufacturer's instruction. pcr-amplification was carried out in multi-prime format. rt-pcr was performed with use of super script iii one- step rt-pcr system with platinum taq (invitrogen, usa) according to the manufacturer's instruction (in μl of reaction mixture: super script iii rt/platinum taq mix- μl; x reaction mix-up to x; primers pmol- μl each; rna- μl each; depc-treated water up to μl). mixture of primers complementary to np and m (aiv), vp (ibdv), s (ibv), np (ndv) gene regions was used for specimen amplification. fluorescent labeling of specimens was carried out by direct embedding of cy -dctp ("amersham", usa) in the process of rt-pcr, the reaction mixture was supplemented in this case with μl of mm cy -dctp. oligonucleotide probes were diluted : with twofold buffer for oligonucleotide printing ("arrayit corporation", usa), in concentration pmol applied on glass slides without support ("sigma", usa) by a method of contact printing with use of nanoprint lm spot-printer ("arrayit corporation", usa). slide contained arrays, where oligonucleotide probes complementary to certain aiv, ndv, ibv, ibdv genome loci were identically immobilized as separate spots μm in diameter. to . μl of pcr-mixture containing cy -cdna hybridization solution was added, the total volume was brought with h o up to . μl and heated in the solidstate thermostat at °c for min, then cooled in ice for min and at once applied onto the microarray. in parallel the oligonucleotide probes on the microarray were denatured by boiling of the slide in h o for min followed by incubation in % ethanol (− °c) for min. after that the slide was dried by centrifugation at g for min. hybridization was performed with use of a frame for subarrays fast® frame ("whatman", usa) for h at °c and stirring at rpm. after hybridization the slide was rinsed in × ssc buffer for min and in × ssc buffer for min to remove unbound molecules of the sample and hybridization buffer. after that the frame was removed and the slide was rinsed with water for min. it was dried by centrifugation at g for min. microarray scanning was carried out by use of innosca-n al scanner ("innopsys", france) at μm resolution and wavelengths nm and nm. the resulted data were processed with the help of mapix ver. . . software ("innopsys", france) and the matrix corresponding to the probe layout on microarray was applied on the obtained pixel image. afterwards the applied array was used to detect probes by intensity of fluorescence with quantitative output with the help of the program module. median fluorescence values of probes minus background signals were considered as effective data. the bigdye terminator cycle sequencing kit was used according to the manufacturer's instructions. the sequencing run was carried out using the -capillary abi prism xl genetic analyser, (applied biosystems). the specificity of the assay was theoretically assessed by evaluating the primers and probes for relevant homologies using the blast tool (https://blast.ncbi.nlm.nih.gov/ blast.cgi). real-time rt-pcr was performed with use of light cycler . manufactured by roche company to detect aiv [ ] , ndv [ ] , ibv [ ] and ibdv [ ] . in assessment of laboratory tests effectiveness trueposi- [ ] . ninety-five percent confidence intervals ( % ci) were calculated according to wilson [ ] . oligonucleotide microarray for rapid detection of aiv, ndv, ibv, ibdv in the result of analysis of nucleotide sequences from ncbi "influenza virus sequence database"genes encoding m and np proteins were chosen for aiv identification. nucleotide sequences of gene encoding np protein were selected for ndv. segment a of vp protein was used for ibdv. nucleotide sequences of gene encoding s protein were chosen for ibv identification. oligonucleotide primers and probes were produced in the course of standard automatic synthesis in dna/ rna synthesizer h- (k&a laborgeraete, germany), their sequences are shown in tables and . the selected oligonucleotide probes were used to develop a microarray for rapid diagnosis of aiv, ndv, ibv, and ibdv. probes were applied on the support by the method of contact printing in nano print lm (arrayit corp., usa). the study has shown that amplification products hybridize on the microarray only in case of obtaining sufficient number of single-chain fluorescently-labeled fragments of np and m (aiv), np (ndv), vp (ibdv), and s (ibv). the electrophoregram has shown pcr products- bp (aiv), bp (ndv), bp (ibv), bp (ibdv) (fig. ) . hybridization on the microarray of the obtained fluorescently-labeled fragments of np and m (aiv), vp (ibdv), s (ibv), np (ndv) genes of the viruses under study resulted in formation on glass slide of stable hybridization complexes with high binding energy and oligonucleotides the sequences of which were complementary to sequences of hybridized np and m of aiv, vp of ibdv, s of ibv, np of ndv gene fragments. for interpretation of the results the microarray layout is shown on fig. . the first two horizontal rows contain universal oligonucleotide probes to np and m genes of aiv, next are probes for detection of vp gene of ibdv and s gene of ibv. last row contains probes for detection of np gene of ndv. in the microarray layout there are identical subarrays arranged into columns and rows containing oligonucleotide probes that are complementary to antisense strand of aiv, ndv, ibv and ibdv genes. the dna-chip was scanned with innoscan al ("innopsys", france) by cy channel activation. the results were interpreted with use of mapix ver. . . software. the signal exceeding the background value was adopted as a positive result. the finding of the study considered reliable if in the course of scanning by cy channel bright fluorescent spots were observed. in the assay of samples the value of specific fluorescence reliably exceeded the value of the background signal (p < . ) (figs. and ) . as figs. and show m and np genes of aiv, vp gene of ibdv, s gene of ibv and np gene of ndv were reliably detected in all specimens. for testing specificity of the method direct sequencing of the pcr-products in abi prism xl genetic analyser, applied biosystems was carried out. the synthesized nucleotide sequences were analyzed using blast program. these nucleotide sequences that are the fragments of np and m (aiv), vp (ibdv), s (ibv), np (ndv) genes were compared with the data of the genbank database. computer analysis has shown the amplified specific pcr-products of aiv, ndv, ibv, ibdv to be parts of their genomes. so, homology of compared sequences confirms specificity of the developed method. the results of the performed experiments prove again that the used fragments of tested genes are highly specific for aiv, ndv, ibv, ibdv. the limit of detection of the assay method is copies of rna. testing of the microarray with use of viruses from the ribsp microbial collection different strains of aiv, ndv, ibv and ibdv were used to test the oligonucleotide microarray. testing was carried out in comparison with real-time rt-pcr (table ) . fifteen different strains of aiv, ndv, ibv and ibdv, diverse in their origin, epidemiological and biological characteristic, were identified correctly with use of dna microarray. diagnostic results of testing dna microarray with use of virus strains from the ribsp microbial collection were comparable to the results of the realtime rt-pcr. sensitivity of the microarray was comparable to the sensitivity of real-time rt-pcr. in large-scale epidemiological studies possibility to analyze concurrently one specimen on many diagnostic probes is extremely important for agent identification. it allows minimizing the time of analysis from several days to several hours. the microarray efficacy in rapid diagnosis of avian viral diseases was evaluated versus virus isolation in embryonated eggs and real-time qpcr for aiv, ndv, ibv and ibdv with use of samples- cloacal swabs and tissue samples from dead birds (table ) . true disease status is determined by the most accurate diagnostic method possible that is called gold standard. in diagnosis of influenza and other avian infections it is virus isolation in chicken embryos followed by identification in hemagglutination inhibition test [ ] , the whole procedure taking from to days. in our study aimed at evaluation of the microarray and real-time rt-pcr effectiveness we used the test of virus isolation in chicken embryos as gold standard. among cloacal swabs analyzed with use of microarray, real-time rt-pcr and virus isolation in embryonated eggs . % of specimens were positive for aiv, . % for ndv. none of cloacal swabs was shown to be ibv and ibdv positive. aiv was detected by the microarray in . % of tissue samples from dead birds, ndv-in . %, ibv-in ndv was detected in . % of samples from great cormorants (phalacrocoracidae family), % from dalmatian pelican (pelecanidae family) as well as in % of samples from broiler chickens of poultry factory "allel agro" (almaty region) and in % of samples from dead domestic chickens in small households of korday district (zhambyl region). ibv and ibdv were detected respectively in . and . % of samples from dead domestic chickens in small households of korday district (zhambyl region). so, samples of were positive for avian infections being diagnosed with use of the dna microarray and virus isolation in embryonated eggs, samples displayed positive results in real-time rt-pcr. diagnostic results of the dna microarray testing with use of experimental specimens from sick and dead birds were comparable to the results of virus isolation in embryonated eggs and real-time rt-pcr. advantage of the dna microarray is simultaneous assay of samples for presence of infections-avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease of birds, while virus isolation in embryonated eggs and real-time rt-pcr allow detecting only one agent in a sample. the results of aiv, ndv, ibv and ibdv detection in clinical specimens by different methods are shown in sensitivity and specificity characteristics of the developed dna microarray and of the real-time rt-pcr as well as positive and negative prognostic values at % confidence interval are shown in table . while virus isolation in embryonated eggs was used as a standard method in our studies the dna microarray demonstrated diagnostic sensitivity equal to . % within % confidence limits . - %) and diagnostic specificity equal to %. high microarray sensitivity is comparable to the diagnostic sensitivity of the realtime rt-pcr equal to . % within % ci limits . - %. diagnostic specificity of the dna microarray and real-time rt-pcr is %. positive predictive values for the dna microarray and real-time rt-pcr are %. negative predictive values are and % respectively. currently, most methods of aiv, ndv, ibv, ibdv and other avian viral agents detection are adapted to specific detection of one agent in a sample. multiplex rt-pcr is successfully used for detection of aiv and its subtypes [ , ] and for diagnosing double infections such as combination of ndv and aiv [ ] . also methods with use of fig. results of scanning labeled cdnas of aiv, ndv, ibv and ibdv strains on microarray. -"a/duck/alberta/ / " (h n ) (aiv); -"a/duck/ germany/ " (h n ) (aiv); -"a/duck/california/ " (h n ) (aiv); -"a/duck/czechoslovakia/ " (h n ) (aiv); -"vinterfild" (ibdv); -"bg" (ibdv); -" " (ibdv); -"koktal" (ibdv); -" / " (ibdv); -"h- " (ibv); -" - " (ibv); -" / " (ndv); -" / " (ndv); -"bor- vgnki" (ndv); -"columba livia/kz/eko/ / " (ndv) multiplex real-time rt-pcr for aiv, ndv and ibv subtypes differentiation have been developed [ ] [ ] [ ] . at present development of a test based on microarray technology for simultaneous detection of aiv, ndv, ibv and ibdv in one sample is important for poultry industry in the republic of kazakhstan. use of microarray improves quality and shortens the analysis duration in molecular diagnosis of infectious diseases and therefore is employed as an independent method in screening for several genes of large numbers of pathology samples [ ] [ ] [ ] . there are biochips for influenza diagnosis that allow screening not only for ha and na, but for m and np genes of influenza a virus [ , ] . in identification of ndv molecular methods with use of oligonucleotides specific to conservative regions of np-gene of ndv were used [ ] . recently vp fig. intensity profiles of hybridization signals of labeled cdnas of strains "a/duck/alberta/ / " (h n ) (aiv); "a/duck/germany/ " (h n ) (aiv); "a/duck/california/ " (h n ) (aiv); "a/duck/czechoslovakia/ " (h n ) (aiv); "vinterfild"(ibdv); "bg" (ibdv); " " (ibdv); "koktal" (ibdv); " - " (ibdv); "h- " (ibv); " - " (ibv); " / " (ndv); " / " (ndv); "bor- vgnki" (ndv); "columba livia /kz/eko/ / " (ndv) gene region of ibdv is successfully used in synthesis of oligonucleotide primers and probes from highly conservative regions for molecular diagnosis [ ] [ ] [ ] [ ] . molecular methods for ibv diagnosis are oriented at using more conservative sequences located in s and s genes of ibv [ , ] . in the proposed microarray probes were developed on the basis of conservative regions of gene fragments encoding np and m (aiv), np (ndv), vp (ibdv), s (ibv) array proteins from genbank database. all viral gene fragments demonstrated high rate of conservatism and therefore the test is universal for detecting aiv, ndv, ibv and ibdv strains. so, high homology of nucleotide sequences of gene regions encoding aiv, ndv, ibv and ibdv array proteins compared to genbank data confirms specificity of the developed microarray for rapid diagnosis of avian influenza, newcastle disease, infectious bronchitis and infectious bursal disease. total analysis duration without time required for the viral rna extraction is - h, and specimens can be simultaneously assayed. duration of the assay with use of the proposed microarray is not longer than in other molecular methods and simultaneous testing of samples for aiv, ndv, ibv and ibdv provides its advantage over other methods. various methods have been developed for the diagnosis of bird infection, such as virus isolation in cell culture, embryonated chicken eggs, or young specificpathogen-free (spf) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (elisa), or immunohistochemistry. all these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. these methods lack the ability to detect low levels of antigens in tissues [ ] [ ] [ ] [ ] [ ] . in the present study field samples ( in total) were used to test effectiveness and reliability of the microarray. nevertheless, positive result of using molecular and biological methods, being very important in emergency cases, should always be confirmed by the method of virus isolation. the results of the study show that diagnostic sensitivity ( . %) and diagnostic specificity ( %) of the dna microarray are comparable with the same of the real-time rt-pcr ( . and %, respectively). diagnostic effectiveness as percentage ratio of true results to the total number of obtained results for the developed dna microarray and real-time rt-pcr was . %. analysis of the obtained data shows that the microarray test for rapid diagnosis of avian infections demonstrates the effectiveness comparable to that of the molecular method real-time rt-pcr and is more rapid and less resource-consuming owing to its ability to detect simultaneously aiv, ndv, ibv ibdv positive samples in the course of one experiment. universality of the test makes it suitable for wide use in veterinary laboratories for prompt detection of avian infections. the developed microarray for rapid diagnosis of avian viral diseases can be used in mass analysis in the system of routine epidemiological surveillance owing to its ability to test one sample for simultaneous detection of aiv, ndv, ibv and ibdv in cases of single and mixed viral infections. at the same time duration of the analysis decreases many times versus classical methods and the proposed scheme of specimen preparation allows conducting assays immediately in small veterinary laboratories thus avoiding transportation of thermolabile rna. the study described in the article "new oligonucleotide microarray for rapid diagnosis of avian viral diseases" was conducted at the research institute for biological safety problems (ribsp), republic of kazakhstan. patent application called "method for rapid diagnosis of avian viral diseases (avian influenza, newcastle disease, infectious bursal disease, infectious bronchitis) on the basis of microarray technology" was registered at the national patent office under the number / . ). in adherence to the virology journal guidelines the ribsp will make freely available any materials and information described in the publication that are reasonably requested by others for the purpose of academic, noncommercial research. this does not alter the authors' adherence to all the virology journal policies on sharing data and materials. consent for publication not applicable. no personal data were collected in the context of this study. re: scientific interest in newcastle disease virus is reviving molecular and structural bases for the antigenicity of vp of infectious bursal disease virus laboratory manual for isolation and identification of avian pathogens infectious bronchitis virus in jordanian chickens: seroprevalence and detection coronavirus avian infectious bronchitis virus review of rapid diagnostic tests for influenza rapid detection and simultaneous subtype differentiation of influenza a viruses by real time pcr detection and subtyping (h and h ) of avian type a influenza virus by reverse transcription-pcr and pcr-elisa rapid multiplex reverse transcription-pcr typing of influenza a and b virus, and subtyping of influenza a virus into h a multiplex reverse transcription-polymerase chain reaction assay for newcastle disease virus and avian pneumovirus (colorado strain) avian influenza virus detection and quantitation by real-time rt-pcr rapid detection and differentiation of newcastle disease virus by real-time pcr with melting-curve analysis development of a real-time taqman rt-pcr assay for 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subtypes among serotype i viruses molecular identification of infectious bursal disease virus strains detection of infectious bursal disease viruses in commercially reared chickens using the reverse transcriptase/polymerase chain reaction-restriction endonuclease assay phylogeny of antigenic variants of avian coronavirus ibv the coronavirus surface glycoprotein detection of infectious bursal disease virus in different lymphoid organs by single-step reverse transcription polymerase chain reaction and microplate hybridization assay point-counterpoint: is the era of viral culture over in the clinical microbiology laboratory comparison of a rapid antigen test with nucleic acid testing during cocirculation of pandemic influenza a/h n and seasonal influenza a/h n comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection not applicable. the present work was supported by grant research project "development and testing of microarray for rapid diagnosis of avian viral diseases", no. /gf (ministry of education and science, republic of kazakhstan). the data-sets analyzed during the current study was available from the corresponding author on reasonable request.authors' contributions ks, nk and vs conceived and designed the experiments. yb, ks, och and nr performed the experiments. ks, nk and vs analyzed the data. ks, ns, as and mo contributed to the work with data analysis and interpretation of results. ks wrote the paper. all authors read and approved the final manuscript. key: cord- -jdq d i authors: meng, qing-wen; zhang, zai-ping; wang, wei; tian, jin; xiao, zhi-guang title: enhanced inhibition of avian leukosis virus subgroup j replication by multi-target mirnas date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: jdq d i background: avian leukosis virus (alv) is a major infectious disease that impacts the poultry industry worldwide. despite intensive efforts, no effective vaccine has been developed against alv because of mutations that lead to resistant forms. therefore, there is a dire need to develop antiviral agents for the treatment of alv infections and rna interference (rnai) is considered an effective antiviral strategy. results: in this study, the avian leukosis virus subgroup j (alv-j) proviral genome, including the gag genes, were treated as targets for rnai. four pairs of mirna sequences were designed and synthesized that targeted different regions of the gag gene. the screened target (i.e., the gag genes) was shown to effectively suppress the replication of alv-j by . - . %. to avoid the generation of escape variants during virus infection, expression vectors of multi-target mirnas were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes). multi-target mirnas were shown to play a synergistic role in the inhibition of alv-j replication, with an inhibition efficiency of viral replication ranging from . - . %. conclusion: the strategy of multi-target mirnas might be an effective method for inhibiting alv replication and the acquisition of resistant mutations. avian leukosis (al) is the general term for a variety of neoplastic diseases of poultry caused by the alpharetrovirus, avien leukosis virus (alv). alv has been classified into subgroups, designated a-j. the subgroup j virus (alv-j) is a relatively new strain of alv that was isolated from dorking fowl in the early s [ ] . alv is an rna virus with a genome of approximately . kb. the proviral genome of alv-j contains three major genes, gag, pol, and env, which encode the viral structural proteins, rna-dependent dna polymerase, and the envelope glycoprotein, respectively. rna interference (rnai) is a simple and effective tool for silencing target genes that involves endogenous or exogenous double-stranded rna (dsrna)-mediated degradation of the specific mrna sequences. the main nucleic acid molecules that induce gene silencing are small interfering rna (sirna) and microrna (mirna), where the sirnas mediate specific mrna degradation, whereas mirna inhibits specific mrna at the translational level. both of these biological processes are considered key methods of modulating host gene expression, and these two molecules are also involved in antiviral and transposon silencing pathways. the rnai strategy has been successfully applied to the inhibition of viral replication. it has been demonstrated that some genes inhibited by sirnas, such as p , vif, nef, tat, and rev, can block human immunodeficiency virus (hiv) replication in cells [ ] . the infection of cells by hiv may be hindered by inhibiting the expression of the hiv receptors cd and cd a, their coreceptors cxcr or ccr , or the virus gag structural protein [ ] . in some studies, transfection of sirna designed to target c virus (hcv) remarkably inhibited the expression of virus-specific proteins and protected cells against hcv rna, in vitro [ , ] . in another study, hepatitis b virus (hbv) replication was successfully inhibited after plasmid expression of hbv sirna transfected into mouse liver [ ] . hu et al. [ ] adopted sirna designed against the alv gag gene and demonstrated significantly reduced virus replication. chen et al. [ ] indicated that alv-b replication was significantly inhibited after knockdown of the alv-b tvb and env genes. although sirnas have been widely used as gene-silencing molecules, the intrinsic drawbacks of sirna methodology have been revealed. off-target effects may be produced where sirna function must be fully and completely complementary to the target sequence; if the virus mutates, sirna will produce off-target effects. other drawbacks include the elicitation of the interferon response and interference with endogenous mirna biogenesis. the unique biogenesis and mechanism of action of mirna do reduce the likelihood of these problems. despite these problems, the rnai strategy remains an attractive option for antiviral therapy and for the functional analysis of genes for several reasons. first, rnai has sequence specificity. second, the application of multi-series rnai can target different genes or sequences simultaneously and hence can minimize the possibility of the virus acquiring mutations that confer resistance. third, rnai can be transmitted from nonpathogenic viruses to pathogenic viruses. finally, due to its sequence specificity, sirna designed against a virus can only inhibit that virus, leaving vaccine strains unaffected. to ensure a high level of rnai gene silencing, multiple mirnas can be designed. these can be transfected into cells using the same transfer medium, and by targeting different sequences, mutation of the target virus, and therefore the probability of evasion from the silencing effect of the mirna is minimized. alv, especially alv-j, brings about enormous economic losses in the poultry industry. the virus has rapidly spread worldwide and transmits both vertically and horizontally. however, chicks are immunologically tolerant to alv infection. unfortunately, to date, no effective vaccine has been developed against alv. in this study, we used rnai technology to inhibit alv replication and screened the effective target sites for their ability to inhibit alv replication at a cellular level. our findings could pave the way for anti-alv gene screening and the development of disease resistance. the sd strains of alv-j were isolated from poultry in shandong, china, and stored at the harbin veterinary research institute (harbin, china), a key state laboratory. the df- cell lines [ ] were provided by zhigao bu at the harbin veterinary research institute (harbin, china). the linearized the pcdna . -gw/emgfp-mir eukaryotic expression vector, escherichia coli top cells and the lipofectamine transfection reagent were purchased from invitrogen (carlsbad, ca, usa). ligase and reverse transcriptase were purchased from takara (dalian, china). the fluorescence quantitative pcr kit was purchased from bioer technology (hangzhou, china). the plasmid extraction kit and the viral rna extraction kit were purchased from shanghai watson biotech (shanghai, china). the goat anti-mouse igg/ fluorescein isothiocyanate (fitc) antibody and the horseradish peroxidase-labeled goat anti-mouse igg were purchased from zhongshan goldbridge biotechnology (beijing, china). the anti-alv-j monoclonal antibody je- was kindly provided by professor qin aijian (yangzhou university, china). all other chemicals were of analytical reagent grade. four pairs of mirnas sequences were designed against the conserved regions of the gag gene (nc- gag) using online software http://rnaidesigner.invitrogen.com/ rnaiexpress/ (table ) . designed mirna sequences were synthesized by shanghai health bioengineering (china). double-stranded oligonucleotide encoding pre-mirna sequence were annealed and inserted into the linearized expression vector, pcdna . -gw/emgfp-mir ( ng/ μl), to construct recombinant plasmids containing the target mirnas, designated mi-gag , mi-gag , mi-gag , mi-gag . all recombinant plasmids have been sequenced to confirm the sequences inserted. two tandem plasmids were constructed based on the mi-gag plasmid backbone. after bgli and xhoi digestion of mi-gag , the mirna sequence of the pol gene (nc- pol), mi-pol (constructed previously, manuscript submitted), was digested with sali and bgli, and ligated to mi-gag . the tandem plasmid consisting of mi-gag and mi-pol was designated mi-g -p . the mi-g -e (env gene: nc- env) and mi-p -e plasmids were constructed in the same way. plasmids were linearized and used to transform e. coli top . positive clones were selected and verified by digestion with xhoi and sali and sequencing. similarly, based on the recombinant plasmid mi-g -p backbone, a tandem plasmid consisting of mi-gag , mi-pol and mi-env was constructed in the same way and designated mi-g -p -e . positive clones were verified by double digestion with xhoi and sali and sequencing. df- cells were cultured in dulbecco's modified eagle's medium (dmem) containing u/ml of penicillin and streptomycin, and containing % (v/v) fetal calf serum (paa gold, austria). twenty-four hours before transfection, cells that had grown to % confluence were used to inoculate cell culture plates at a density of - × . when the cells had grown to % confluence, they were transfected with the rnai expression plasmid using lipofectamine . six hours later, the culture fluid was changed dmem and serum containing % fetal calf serum, without antibiotics. after incubation at °c and % co for h, the transfection efficiency was determined by the expression of green fluorescent protein observed using fluorescence microscopy. transfected cells were inoculated with % tissue culture infectious doses (tcid ) of alv-j. the culture medium was obtained h after viral infection, and the inhibitory effect was detected by an indirect immunofluorescence assay (ifa), western blotting, and real-time pcr. in this study, the negative control group that was not administered plasmid, the null vector control group and the test groups were randomized for analysis. strain sd was inoculated into df- cell culture bottles, freeze-thawed three times and then centrifuged at × g and °c for min. the supernatant was collected and -fold serially diluted to - and then inoculated into -well plates along with df- cells. four parallel well were created in each gradient. after incubation for days, cells were fixed by cooling in methanol at - °c for min, and then washed three times with phosphate buffered saline containing . % tween (pbst) for min each. alv-j specific monoclonal antibody je- was diluted : and μl was added to each well. the cells were incubated at °c for h and washed with pbst three times, for min each. the secondary antibody, goat anti-mouse igg/fitc, was added at a dilution of : , and the cells were incubated at °c for min away from the light. the cells were then washed with pbst five times, for min each. the number of fluorescent cells per well were counted using a fluorescence microscope, and the tcid was calculated using the reed-muench method [ ] . therefore, the toxic potency of the virus was determined to be . tcid /ml. virus strain sd was used to inoculate df- cells transfected with recombinant plasmid using the method described above. after days incubation, cells were treated with the fluorescent secondary antibody, rhodamine-labeled goat anti-mouse igg/tritc, as described above. the results were photographed using a fluorescence microscopy. at about h after transfection with the mirna recombinant plasmid, df- cells were collected and the residual supernatant fluid was washed with pbs. total protein was extracted using the total protein extraction kit (best-bio, china) and quantitated using a spectrophotometer. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page; %) was performed using a sample volume of μg per well. samples were transferred to nitrocellulose membrane after separation, blocked at °c overnight and then washed with pbst three times, for min each. alv-j-specific monoclonal antibody je- was added and the cells were incubated at °c for h and mouse anti-chicken glyceraldehyde- -phosphate dehydrogenase (gapdh) monoclonal antibodies were added and incubated at room temperature for h, followed by three washes with pbst for min each. horseradish peroxidase-labeled anti-mouse secondary antibody was added and cells were incubated at °c for min. after washing with pbst five times, min each wash, the cells were stained with , ' diaminobenzidine (dab, mg, ml tbs, . ml % h o ) and then scanned using the gel imaging system alphaimager hp. using sequences of the alv gag genes published in the genbank database (nc- ), pairs of primers were designed using the oligo primer design software to amplify the conserved regions of the gene. additionally, a pair of alv-j specific primers was also synthesized. two pairs of target genes and the b-actin gene were amplified by pcr using the sd cdna as a template ( table ). the cdnas amplified using the gag, alv-specific, and b-actin primers were cloned into vector pmd- -t and used to transform competent e. coli dh α cells. positive clones were isolated and verified by colony pcr and sequencing, and designated pmt-g and pmt-alv, respectively. the concentration and purity of verified plasmids were determined according to the formula: copy number = (mass/molecular weight) × . × . extracted plasmids were then ten-fold serially diluted and used as temples for quantitative pcr amplification and delineation of quantitative pcr standard curves. total rna was extracted from viruses and cells h after the infection of df- cells with alv-j using trizol, according to the manufacturer's instructions. rna concentration and purity was determined by measuring optical density (od) at wavelengths of and nm using a standard spectrophotometer. the od / od ratios were more than . for all samples. quantitative pcr was performed in a lightcycler real-time pcr system, for the gag and alv-specific genes using the sybr green kit (sybr ® premix ex taq™, takara, dalian, china) and the primers are listed in table . amplification was carried out in a μl reaction mixture containing μl sybr ® premix ex taq™ ×, . μm concentration of each primer, μl cdna. the reaction procedure was °c s, followed by cycles at °cfor s and °c for s. the number of target genes in different samples was determined according to the standard curve after the reaction. meanwhile, the copy number of the b-actin gene was also determined by quantitative pcr. to confirm specific amplification, melting curve analysis was performed. experiments were repeated three times and values were expressed as means ± standard deviation (sd). the ttest was performed using the spss . statistical software (version . ; spss, usa). differences were considered statistically significant when p < . . as shown in figure a and b, ifa revealed that the recombinant plasmids, mi-gag and mi-gag , significantly reduced the fluorescence intensity. the tandem plasmids, mi-g -p , mi-g -e , mi-p -e and mi-g -p -e , also significantly inhibited the fluorescence intensity (figure a and b) compared with the negative control group. however, no significant differences were detected among other groups compared with the negative control group and the null vector group. western blot analysis of the alv-j envelope glycoprotein je- is an anti-alv-j envelope glycoprotein monoclonal antibody that specifically identifies a protein with a molecular weight of - kda. as detected by western blot analysis (figure a, b) , the recombinant mi-gag and mi-gag plasmids decreased alv-j envelope glycoprotein expression significantly, while there were no significant differences determined between the remaining groups and the negative control or null vector groups. transfection of the tandem plasmids into df- cells significantly decreased the expression of the alv-j envelope glycoprotein, indicating that alv-j could inhibit plasmid replication. expression of the gag gene in cells transfected with plasmids mi-gag and mi-gag differed significantly from the negative control group and the null vector control group (p < . ), while expression in cells transfected with plasmids mi-gag and mi-gag did not differ significantly from that in the negative control and null vector control groups (p > . ) ( table ). the use of tandem plasmids indicated significant inhibition of the expression of the alv-j envelope glycoprotein in df- cells ( - . %), and the rate of inhibition was highest with plasmid mi-g -p -e ( . %) ( table ). accumulating evidence suggests that rnai can inhibit viral replication in vivo and in vitro. rnai technology have been applied in numerous studies including those for hbv [ ] , hcv [ ] , hiv- [ ] , influenza virus a [ ] , aphthovirus of cattle [ , ] , and severe acute respiratory syndrome (sars) virus [ ] infections. rnai has been used to suppress the replication of herpesviruses, including murine herpesvirus [ ] , herpes simplex virus- [ ] , human cytomegalovirus [ ] , and duck herpesvirus [ ] . the alv-j genome contains a gene arrangement of ltr-leader-gag-pol-env-ltr. the gag and pol genes are highly conserved, sharing - % homology in alv-j, in contrast to subgroups a, c, and d. the pol gene mainly encodes the reverse transcriptase (rt; p ) and viral integrase (in; p ). rt is responsible for the production of proviral dna using viral rna as a template, while in is involved in the integration of proviral dna into the host genome. the pol gene is necessary for reverse transcription of the rna genome and the generation of viral dna, and it is a key to the insertion of the viral genome into the host genome. rnai has been applied to the inhibition of alv replication by a number of research groups. chen et al. [ ] successfully inhibited alv-b replication induced by a retroviral vector by targeting the mirna of the alv-b env gene and its receptor encoded by the tvb gene. by constructed an mirna expression vector targeting the alv-j gag gene, we demonstrated that mi-gag and mi-gag could significantly reduce the expression of target gene mrna and envelope glycoprotein at a cellular level, with the highest inhibition rate of . % being observed with mi-gag . these results showed that mirna expression could inhibit the duplication of the target gag gene, with mi-gag having the highest inhibitory effect. accordingly, the mirna of the gag target genes could successfully inhibit alv-j replication. thus, the successful construction of a eukaryotic expression vector would contribute to the selection and propagation of an anti-alv related gene. in previous studies, rnai strategies have been successfully employed experimentally to inhibit virus replication. hu et al. [ ] showed that by electroporation into chicken embryos, sirna containing alv gag sequences effectively slowed down virus propagation. in the current study, the mirna target gag was selected that resulted in significant inhibitory effects on alv-j replication. these mirnas were grouped in pairs or together, and the tandem multi-target mirnas target mi-g -p , mi-g -e , mi-p -e , and mi-g -p -e were co-expressed, reducing the probability of evasion from the silencing effect of the mirna due to virus mutation. when multiple mirnas were expressed simultaneously, gene silencing was shown to be more effective. tandem plasmids were transfected into df- cells and then infected with alv-j. ifa, western blotting, and quantitative pcr were used to evaluate the inhibiting effects on alv-j replication at the cellular level. the results verified that all of the tandem plasmids could effectively inhibit the replication of alv in df- cells, with an inhibition efficiency of - . %. the enhanced inhibitory effects conferred by the multi-target mirna expression plasmids demonstrated that the multi-target mirnas could inhibit alv synergistically. these studies successfully identified targets capable of inhibiting the replication of alv-j and are likely to be good candidates for the development of mirna-based vaccines. the current study showed that the strategy of using multi-target mirnas might be an effective method for inhibiting viral replication and for the acquisition of resistant mutations. detecting avian leukosis virus subgroup j using tissue chip and immunohistochemical technology molecular strategies to inhibit hiv- replication expression of small interfering rnas targeted against hiv rev transcripts in human cells the efficacy of sirna against hepatitis c virus is strongly influenced by structure and target site accessibility inhibition of hepatitis c virus infection in cell culture by small interfering rnas inhibition of hepatitis b virus in mice by rna interference inhibition of retroviral pathogenesis by rna interference inhibition of avian leukosis virus replication by vector-based rna interference the df chicken fibroblast cell liner: transformation induced by diverse oncogene and cell death resulting from infection by avian leukosis viruses a simple method of estimating fifty percent endpoints simultaneously inhibition of hiv and hbv replication through a dual small interfering rna expression system interfering with hepatitis c virus rna replication potent and specific inhibition of human immunodeficiency virus type replication by rna interference rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcriptioin rna interference targeting vp inhibits foot-and mouth disease virus replication in bhk- cells and suckling mice cross-inhibition to heterologous foot-and-mouth disease virus infection induced by rna interference targeting the conserved regions of viral genome attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rna-dependent rna polymerase inhibition of gammaherpesvirus replication by rna interference short interfering rna-mediated inhibition of herpes simplex virus type gene expression and function during infection of herpes simplex virus type gene expression and function during infection of human keratinocytes inhibition of human cytomegalovirus replication by small interfering rnas inhibition of anatid herpes virus- replication by small interfering rnas in cell culture system submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution authors' contributions qwm conceived the study, participated in its design and coordination, and finalized the manuscript in its final form. zpz performed the experiments. ww carried out the statistical analyses and participated in drafting of the manuscript. jt carried out the replication studies. zgx carried out the quantitative pcr. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -a eh dkb authors: kwofie, theophilus b; anane, yaw a; nkrumah, bernard; annan, augustina; nguah, samuel b; owusu, michael title: respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: a eh dkb background: acute respiratory tract infections are one of the major causes of morbidity and mortality among young children in developing countries. information on the viral aetiology of acute respiratory infections in developing countries is very limited. the study was done to identify viruses associated with acute lower respiratory tract infection among children less than years. method: nasopharyngeal samples and blood cultures were collected from children less than years who have been hospitalized for acute lower respiratory tract infection. viruses and bacteria were identified using reverse transcriptase real-time polymerase chain reaction and conventional biochemical techniques. results: out of patients recruited, ( . %%, %ci: . % to . %) were positive for one or more viruses. respiratory syncytial virus (rsv) was detected in ( . %, %ci: . % to . %) patients followed by adenoviruses (adv) in ( . %, %ci: . % to . %), parainfluenza (piv type: , , ) in ( . %, %ci: . % to . %) and influenza b viruses in ( . %, %ci: . to . ). concomitant viral and bacterial co-infection occurred in two patients. there were no detectable significant differences in the clinical signs, symptoms and severity for the various pathogens isolated. a total of . % ( / ) of positive viruses were detected during the rainy season and respiratory syncytial virus was the most predominant. conclusion: the study has demonstrated an important burden of respiratory viruses as major causes of childhood acute respiratory infection in a tertiary health institution in ghana. the data addresses a need for more studies on viral associated respiratory tract infection. acute respiratory infections (ari) are one of the major causes of morbidity and mortality in young children throughout the world especially in developing countries [ , ] . data from who estimated the burden of ari at , , disability-adjusted life years (dalys) and . million deaths in [ ] . similar report from a metaanalysis study demonstrates that throughout the world . million ( % ci . - . million) children died from ari in , % of them in africa and southeast asia [ ] . a further systematic analysis also estimated . million (uncertainty range: . million - . million) deaths of children worldwide in as due to ari [ ] . majority of acute lower respiratory tract infections (alrti) in developed countries have been reported to be often due to viral pathogens of which most common are rsv, piv, influenza viruses, adv, human coronaviruses and bocaviruses [ ] [ ] [ ] . on the contrary, information on these viruses in developing countries is limited probably due to paucity of modern diagnostic molecular techniques. these infections are therefore treated unsuccessfully with antibiotics based on suspicion of bacterial causes [ ] . apart from the public health concern of nosocomial infections that are associated with viral respiratory infections [ , ] , significant costs derived from long duration of hospitalization and several healthcare visits could also aggravate the poor socio-economic status and increased child mortality in developing countries including ghana. lessons from the outbreak of the severe acute respiratory syndrome (sars) epidemics which resulted in the death of individuals [ ] and the recent emergence of a novel swine flu pandemic emphasize the risk posed by respiratory viral infections in humans [ ] . this study was done to determine the burden of respiratory viruses among children hospitalized at the komfo anokye teaching hospital for acute lower respiratory illness using the real time polymerase chain reaction (rt-pcr). this was a hospital based cross-sectional study of children less or equal to five years and hospitalized for acute lower respiratory tract illness. the study was performed at the children's ward of the komfo anokye teaching hospital (kath), ghana from january to december . kath is approximately a thousand bed tertiary medical facility located in ashanti region, kumasi, the confluence of the transportation network in the central part of ghana. its position makes it the most accessible tertiary and referral medical facility in ghana attending to a population of over . million in the ashanti region and beyond [ ] . to have a year round incidence of the various viral agents, recruitment was done throughout the year . screening and recruitment were started at the beginning of every week till two or three patients were recruited. after a maximum of three patients have been obtained it was suspended till the beginning of the next week. this cycle was repeated till the maximum of patients were recruited for each month. at the recruitment station all patients arriving at the unit were screened but only those less than five completed years were recruited. patients recruited into the study should have features of severe pneumonia or very severe pneumonia as defined by the who-imci (world health organisation integrated management of childhood illness) protocol [ , ] . severe pneumonia was said to be present if the child has a history of cough and/ or difficult breathing of less than weeks duration, with lower chest wall recession. severe pneumonia in addition to cyanosis and/or inability to feed or drink was classified as very severe pneumonia. a child was recruited into the study only after the guardian or parents had consented after the objectives of the study had been explained in english or the local dialect. a standardized case record form was then used to record the history of the illness as well as the presenting clinical features of pneumonia. five ( ml) of blood sample was then taken into ml of brain heart infusion broth (bhib) and quickly sent to the bacteriological laboratory for culture. nasopharyngeal specimens were also taken using the nasopharyngeal flocked swab (copan, italy). the swab was gently inserted up the nostril towards the pharynx until resistance was felt and then rotated times to obtain epithelial cells. it was then withdrawn and put into . ml phosphate buffered saline (x ). the samples were transported on ice to the laboratory within few hours of collection. they were then vortexed, transferred into . ml eppendorf tubes (eppendorf, germany) and kept at - °c for polymerase chain reaction (pcr). both samples were taken by experienced prior trained nurses of the children ward. children were excluded if both samples could not be obtained for any reason. viral nucleic acids were extracted from samples using qiaamp viral mini kit (qiagen, germany) according to the manufacturers' instruction [ ] . rt-pcr amplification was performed for the detection of adv, rsv, influenza a (flu a), influenza b (flu b), parainfluenza (piv ), parainfluenza (piv ) and parainfluenza (piv ) using the light cycler version . (roche, germany). primers highly specific to each of the viruses were used ( table ). the reaction mixture for rna viruses was made up of μl of rna extract, μl deoxynucleotide (dntp) triphosphate ( mm) (qiagen), μl bovine serum albumin (bsa)( mg/ml)(qiagen), . μl qiagen one step rt-pcr buffer x (containing . mm mgcl ) (qiagen), μl qiagen one step enzyme mix, μl each of sense and antisense primer( μm each), . μl probe and . μl of rnase free water. dna virus reaction mixture was made up of μl pcr buffer ( x), μl of dntp mix ( mm each), μl of mgcl ( mm), . μl of hot star taq dna polymerase, . μl of probe, μl of bsa ( mg/ml) and μl each of sense and antisense primers. the pcr conditions for piv, rsv and influenza viruses consisted of an initial reverse transcription at minutes for °c, taq dna polymerase activation at °c followed by forty five ( ) cycles of denaturation at seconds for °c and annealing at seconds for °c. the initial reverse transcription step was omitted in the case of adv. for each batch of tests that were run, rnase free water (qiagen, germany) was used as negative control and in vitro transcribed gene of the various viruses was used to determine the detection limit of the assay as positive control. the in vitro transcripts were prepared in our collaborative institution (bonn institute of virology, bonn, germany) and transported to ghana via cooling chain. the in vitro transcripts were prepared by amplifying the genes of interest of the viruses with specific set of primers. the products were ligated to the pcr . vector using the topo ta cloning kit (invitogen corp, carlsbad, ca) and transformed into competent e.coli cells. after overnight incubation, desirable colonies were selected and purified using qiamp spin-miniprep kit (qiagen, germany). pcr was performed on the purified product and rna transcripts were then synthesized from the lowest dilution (with clear band) using ambion megascripts t kit (invitogen corp, calsbad, ca). the final products were purified using rneasy mini kit (qiagen, germany) and the concentrations of the transcripts were determined using a spectrophotometer (themoscientific, u.s.a). the copy numbers of the rna transcripts were determined following the method of fronhoffs [ ] . ten-fold dilutions of the rna transcripts were prepared in carrier rna-rnase free water ( μg of carrier rna in ml of rnase free water) and tested. the detection limit was determined as the last dilution after which all other replicates gave negative results. the detection limits were respiratory syncytial virus (rsv): copies/μl, piv : - copies/μl, piv : copies/μl, piv : copies/μl, influenza a: copies/μl and influenza b: copies/μl. to determine the specificities, our assays were tested on other different viruses and they turn out negative. bacterial isolates were identified using conventional biochemical methods including urease and indole production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars. the biochemical media used included simon's citrate medium, urea and triple sugar iron agar (tsi). coagulase tests were performed for all staphylococci organisms. data obtained was double entered into a spreadsheet database prepared with microsoft ® excel. it was then compared and cleaned for abnormal wrongful entries. statistical analysis was done using stata se statistical software version . (texas, usa) after the data had been imported. categorical variables such as age groups and their association with respiratory agents were analyzed using the fischer's exact test. continuous variables were expressed as medians with their inter-quartile ranges. a non-parametric k-sample test on the equality of medians was used to evaluate the differences in the medians of the various subgroups of the continuous variables. for all analysis done, a p-value of less than . was considered statistically significant. the study protocol was approved by the six ( . %) out of children less than six months of age were found to be positive for viral infection ( table ). the highest number of children infected were between months and years, the proportions infected for the various age groups were however not significantly different. clinical presentation of patients were compared to general pathogens identified (table ) and the individual viral pathogens (table ) . generally, there were no detectable significant differences in the clinical presentation as well as the gender and duration of illness of patients for the various pathogens isolated. the study also investigated the seasonal variation of viruses. rsv infections were detected throughout the year however the peak infection rate occurred during the minor rainy seasons (october) (figure ). adenovirus infections on the other hand had two main peaks occurring in the month of april and october. viral agents play an important role in acute lower respiratory infections and may herald the onset of pneumonia caused by secondary bacterial infections [ ] . although information on the causes of respiratory illness in tropical countries is very scanty, available data indicates about one -third of the cases of respiratory tract infections are due to viruses [ ] . the present study identified viruses in . % of patients hospitalized for alrti with rsv being the most predominant. the overall prevalence is comparable to previous studies done in other developing countries [ ] and the predominance of rsv is in accordance with the assertion that this virus is the single most frequent lower respiratory tract pathogen in infants and young children worldwide [ ] [ ] [ ] . adenoviruses, the second highest viruses detected in this study have been reported to be responsible for - % of lower respiratory tract infections with the highest rate occurring in younger children [ ] [ ] [ ] [ ] . our study similarly recorded a . % detection rate of adenoviruses however there was no statistical difference in the age specific prevalence. this could possibly be due to the few number of younger children (less than one year) enrolled in this study. this study generally recorded higher cases of tachypnoea, chest recessions and very severe pneumonia in rsv compared to adenovirus infected patients but the differences were not statistically significant. although similar clinical presentations have been found to be associated with rsv [ , ] , our small sample size could account for our inability to detect these differences. among patients who were poorly fed, those without viral or bacterial infections were found to be higher (p = . ). poor feeding and malnutrition has generally been associated with less risk for respiratory viral infection in developing countries [ ] [ ] [ ] however the possibility of increased mortality could occur especially when bacterial infections are involved. further case controlled studies are therefore needed in developing countries to investigate this observation there have been fewer studies of piv infections in developing countries and most of them do not differentiate the subtypes of the viruses. our study recorded a . % prevalence of piv infections with the predominant type being piv- . similar hospital based studies have been reported in other developing countries [ ] [ ] [ ] [ ] . piv infections have been strongly associated with croup [ , ] . the present study recorded no case of croup among piv patients. viral associated bacteremia was detected in two patients ( . %). low rate of secondary bacterial infections has generally been reported in developing countries with the isolation rate varying between and % [ ] [ ] [ ] . studies in some developed countries however reported the converse [ , ] probably due to the higher sensitivity of the bacteria identification techniques used. the blood culture identification system used in this study may be limited in the detection of bacteria associated with lower respiratory tract infection. perhaps the detection rate could have increased if the present study had identified bacterial pathogens from nasopharyngeal aspirates or washings as reported by hishinki et al. [ ] . the isolation of staphylococcus aureus in an rsv positive patient in the present study was similar to studies reported by berman et al., [ ] and cherian et al., [ ] . in their case, the bacterium was associated with fatality in the children. the present study observed that all subjects enrolled in this study were treated empirically with antibiotics in accordance with the paediatric emergency treatment protocol for managing severe and very severe pneumonia. similar occurrences have been reported where patients were treated unsuccessfully with multiple antibiotics [ ] . policy makers may therefore consider reviewing the clinical algorithm for patient management as the economic cost both to the health service and the patient's household derived from the use of antibiotics could be enormous. the period of this study experienced major rainfalls in the months of may to july and minor rainfalls in the month of september to october. the month of october recorded the highest detection of viral infections ( . %; / ) with the commonest being rsv. this phenomenon has been reported in other developing countries [ , ] . the occurrence of rsv infections in the rainy and cold season could be due to overcrowding as a result of populations staying more to their homes. more studies are however needed to define the seasonality of respiratory viruses in tropical countries. our study had some limitations which include underestimation of the overall prevalence of respiratory viruses since we did not test for coronaviruses, human metapneumovirus, bocaviruses and rhinoviruses which have also been reported in hospitalized patients. also the negative results recorded for influenza a and piv and the low prevalence of influenza b in our samples could be due to the low sensitivity of the assay for these viruses. the limit of detections of influenza a, influenza b and piv are copies/μl, copies/μl and copies/μl respectively as such viral copies below these limits of detections could be missed by our assay. this study has demonstrated that respiratory viruses are associated with a considerable number of hospital admissions in ghana. hospitalized children presenting with symptoms of alrti may not necessarily need treatment with antibiotics but rather antiviral drug options could be explored. the importance of further cross-sectional and longitudinal studies of viral-associated respiratory infections among out-patients and healthy populations cannot be over emphasized. this will contribute immensely to overcoming the paucity of data on the importance of respiratory associated viruses to the burden of disease and the morbidity and mortality of under five children. acute respiratory infections are the leading cause of death in children in developing countries estimates of worldwide distribution of child deaths from acute respiratory infections world health organisation: burden of disease in dalys by sex and mortality stratum in who regions, estimates for . the world health report global, regional, and national causes of child mortality in : a systematic analysis progress in the surveillance of respiratory syncytial virus (rsv) in europe development of three multiplex rt-pcr assays for the detection of respiratory rna viruses population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children risk of secondary bacterial infection in infants hospitalized with respiratory syncytial viral infection the clinical spectrum of respiratory syncytial virus disease in the gambia an outbreak of acute respiratory disease in trinidad associated with para-influenza viruses newly discovered coronavirus as the primary cause of severe acute respiratory syndrome emergence of a novel swine-origin influenza a (h n ) virus in humans komfo anokye teaching hospital, centre of excellence integrated approach to child health in developing countries integrated management of childhood illness by outpatient health workers: technical basis and overview. the who working group on guidelines for integrated management of the sick child qiagen: protocol: purification of viral rna (spin protocol) a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction epidemiology of acute respiratory infections in children of developing countries. review of infectious diseases viral respiratory infections and their role as public health problem in tropical countries (review) respiratory syncytial virus infection in tropical and developing countries update: respiratory syncytial virus activity-united states, - season epidemiology and aetiology of acute bronchiolitis in hong kong infants viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in - frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real time pcr assay adenovirus pneumonia in infants and factors for developing bronchiolitis obliterans: a -year follow-up epidemiology and clinical presentation of respiratory syncytial virus infection in a rural area of southern mozambique clinical presentation and severity of viral community-acquired pneumonia in young nepalese children the etiology of pneumonia in malnourished and well-nourished gambian children respiratory syncytial virus infections in malnourished children viruses associated with acute lower respiratory tract infections in children from the eastern highlands of papua new guinea ( - ) epidemiology of acute respiratory tract infections among young children in kenya. review of infectious diseases viral etiology and epidemiology of acute respiratory infections in children in etiology of acute lower respiratory tract infections in gambian children: i. acute lower respiratory tract infections in infants presenting at the hospital viral etiology of respiratory infections in children under years old living in tropical rural areas of senegal: the evira project respiratory syncytial virus and parainfluenza virus parainfluenza viruses bronchiolitis in tropical south india etiology of acute respiratory infections in children in tropical southern india. review of infectious diseases diagnosis of childhood pneumonia in the tropics association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients acute lower respiratory tract illnesses in cali, colombia: a two-year ambulatory study seasonal variation in respiratory syncytial virus infections in children in evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children simultaneous detection of influenza viruses a and b using real-time quantitative pcr respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana the study was partly supported by the komfo anokye teaching hospital (ghana) and the kumasi center for collaborative research in tropical medicine (ghana). we will like to thank all directorate of child health doctors, nurses and patients at the komfo anokye teaching hospital. we also thank the bonn institute of tropical medicine (germany) for supporting us with laboratory materials. authors' contributions yaa, aa and bn co-worked on the data collection and contributed to laboratory analysis of the samples. sbn performed statistical analysis of the data and contributed to writing of the manuscript. tbk planned, initiated the study and contributed to writing of the manuscript. ow carried out the laboratory analysis of the samples and contributed to writing of the manuscript and interpretation of the data. all authors have read and approved the manuscript. the authors declare that they have no competing interests. key: cord- -we pveus authors: ehlen, lukas; tödtmann, jan; specht, sabine; kallies, rené; papies, jan; müller, marcel a.; junglen, sandra; drosten, christian; eckerle, isabella title: epithelial cell lines of the cotton rat (sigmodon hispidus) are highly susceptible in vitro models to zoonotic bunya-, rhabdo-, and flaviviruses date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: we pveus background: small mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. however, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. the cotton rat (sigmodon hispidus) is a new world rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens. methods: a method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. susceptibility to members of zoonotic bunya-, rhabdo-, and flaviviridae, in particular rift valley fever virus (rvfv), vesicular stomatitis virus (vsv), west nile virus (wnv), and tick-borne encephalitis virus (tbev) was tested. furthermore, novel arthropod-derived viruses belonging to the families bunya-, rhabdo-, and mesoniviridae were tested. results: we successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. cells were shown to be interferon-competent. viral infection assays showed high-titre viral replication of rvfv, vsv, wnv, and tbev, as well as production of infectious virus particles. no viral replication was observed for novel arthropod-derived members of the bunya-, rhabdo-, and mesoniviridae families in these cell lines. conclusion: in the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. these cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species. infectious diseases are a major threat to human health and remain among the leading causes of death and disability worldwide [ ] . in the last decade, a variety of viruses such as ebola virus, hendra virus, nipah virus, west nile virus (wnv), and severe acute respiratory syndrome (sars)and middle east respiratory syndrome (mers)-coronaviruses have emerged or re-emerged, all of which are of zoonotic origin [ ] [ ] [ ] [ ] . there have been a large number of novel, potentially zoonotic viruses that have been shown to be associated with small mammals, especially those of the orders chiroptera and rodentia, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the isolation and propagation of these novel viruses has been unsuccessful in most instances, which limits further evaluation of their zoonotic risk. upon characterizing these novel viruses, it has become clear that most available animal models such as the domestic mouse or rat are of limited use, as they do not reflect the evolutionary conserved pathogen-host interaction that is a key trait of many reservoir-restricted viruses. in light of the large species range in which novel and potentially zoonotic viruses have been discovered, there remains a need for suitable in vitro models to understand virus-host interactions, interspecies spillover, and general viral pathogenicity [ ] . additionally, many of the natural reservoir hosts are protected or cannot be held in captivity, which limits in vivo studies in relevant hosts. therefore, species-specific cell culture models may serve as acceptable surrogates [ ] [ ] [ ] [ ] . the cotton rat (sigmodon hispidus) is a unique example of a rodent species that is a well-established animal model to study viral pathogenesis and is also associated with a large range of zoonotic viruses in the wild [ ] [ ] [ ] . experimental studies in cotton rats have been performed for a large variety of human viruses, including important respiratory pathogens such as influenza or parainfluenza viruses, respiratory syncytial virus, and human metapneumovirus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, in the wild, cotton rats are associated with a variety of known or potential zoonotic viruses, such as classical rodent-borne viruses from the genera hantavirus and arenavirus, as well as members of the family flaviviridae, such as wnv and st. louis encephalitis virus (slev) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to evaluate whether the broad viral susceptibility seen in both animalmodel and wild cotton rats was also reflected in in vitro cell culture models, we generated continuous cell lines from the respiratory and renal tracts of a cotton rat, and assessed their use for virus replication studies of known and potentially novel zoonotic viruses. tissues from a laboratory-bred -month-old male cotton rat (s. hispidus) were kindly provided by the institute for medical microbiology, immunology and parasitology (immip), university of bonn medical centre, bonn, germany. ethical clearance was obtained from the respective authorities (no. az - . . . . ). the trachea and both kidneys of the euthanized cotton rat where removed in toto. all subsequent steps were then performed under sterile conditions using a laminar flow hood. briefly, organ specimens were cleaned from surrounding tissue and then either sliced or roughly chopped with a sterile blade. tissue slices were placed in -well cell culture plates at °c in primary cell media. for tracheal cells, airway epithelial cell growth medium was used containing the following supplements: . ml/ml bovine pituitary extract, ng/ml recombinant human epidermal growth factor, μg/ml recombinant human insulin, . μg/ml hydrocortisone, . μg/ml epinephrine, . ng/ml triiodo-l-thyronine, μg/ml human holotransferrin, and . ng/ml retinoic acid (promocell, heidelberg, germany). for kidney cells, renal epithelial cell growth medium was used containing the following supplements: . ml/ml foetal calf serum (fcs), ng/ ml recombinant human epidermal growth factor, μg/ml recombinant human insulin, ng/ml hydrocortisone, . μg/ml epinephrine, pg/ml triiodo-l-thyronine, and μg/ml human holo-transferrin (promocell). both media were supplemented with % penicillin/streptomycin (life technologies gmbh, darmstadt, germany), . % of ofloxacin (tarivid, sanofi-aventis, frankfurt, germany) and % amphotericin b (paa, pasching, austria) to avoid bacterial and fungal contamination during primary cell isolation and growth. after the outgrowth of primary cells from organ specimens, the medium was changed every days, and cell outgrowth was regularly observed. when nearly confluent, cells were immortalized by lentiviral transduction of the large t antigen of sv as described previously [ , , ] . after immortalization, cells were passaged and stock-frozen until further use. all cell cultures were genotyped by polymerase chain reaction (pcr) amplification and sequencing of the mitochondrial cytochrome c oxidase subunit i and cytochrome b oxidase subunit i genes [ , ] . to obtain single cell clones, cells were subcloned by end-pointlimiting dilution and adapted to dulbecco's modified eagle's medium (dmem) (paa, cölbe, germany) with . g/l glucose (paa), supplemented with % fcs (paa), mm l-glutamine, mm sodium pyruvate (paa), nonessential amino acids (neaa), % penicillin/streptomycin ( x concentrate contains , units/ml penicillin and mg/ml streptomycin) (life technologies), and % amphotericin b as described previously [ , ] . cells were seeded on glass slides, and were washed the next day with pbs and fixed with acetone-methanol ( : ) for min. then, the acetone-methanol was removed and cells were washed again with pbs. each slide was subsequently incubated overnight at °c with μl primary mouse monoclonal antibodies against pan-cytokeratin (abcam ab , cambridge, uk) and rabbit polyclonal antibodies against zonula occludens- (zo- mid) (invitrogen - , carlsbad, ca, usa) diluted : in pbs. cells were washed and then incubated for min at °c with μl cyanine (cy )-labelled donkey-antimouse and cy -labelled donkey-anti-rabbit secondary antibodies (dianova, hamburg, germany) diluted : in pbs. cells were washed and then nuclei were counterstained with dapi diluted at : in pbs for min. all images were obtained with a motic axiovision microscope (zeiss, jena, germany). immortalized s. hispidus cells were seeded in -well plates at a density of × cells/ml and grown in dmem containing % fcs and supplements as described above. the following day, cells were infected with vesicular stomatitis virus (vsv) strain indiana or rift valley fever virus (rvfv) clone at multiplicity of infections (mois) of . and . for both viruses. cells were infected with wnv strain new york or tick-borne encephalitis virus (tbev) strain k with mois of . and . . infectious units of the viral stocks and in the supernatant at the end of each experiment were determined by plaque-assays with avicel overlays for rvfv and vsv as described previously [ ] , and with agarose overlays for wnv and tbev as described previously [ ] . for virus infection experiments, the medium was removed and cells were inoculated with virus diluted in optipro serum-free medium (life technologies) for h at °c. then, cells were washed twice with pbs. growth medium was added and supernatants were harvested , and h after infection (hpi) for vsv; , and hpi for rvfv and , , and hpi after infection for wnv and tbev. all virus infection experiments were performed in three individual replicates. viral rna was extracted from cell culture supernatants with the nucleospin rna virus kit according to the manufacturer's instructions (machery-nagel, düren, germany). pcr was performed using the superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen). cycling conditions for vsv and rvfv quantitative reverse-transcription (qrt)-pcr were as follows: reverse transcription for min at °c, initial denaturation for min at °c, and cycles of denaturation for s at °c and primer annealing/elongation for s at °c. cycling conditions for wnv qrt-pcr were as follows: reverse transcription for min at °c, initial denaturation for min at °c, and cycles of denaturation for s at °c and primer annealing/ elongation for s at °c. qrt-pcr was carried out using the lightcycler real-time pcr system (roche, basel, switzerland). primers and probes are available upon request. to test the susceptibility of the s. hispidus cell lines to a variety of novel arthropod-derived viruses, cells were seeded in -well plates at a density of × cells/ml. the following day, cells were infected with a titrated c / cells-generated virus stock of ferak [ ] , moussa [ ] , or cavally [ ] virus at an moi of . . after infection, cells were observed daily for the presence of cytopathic effects (cpe). supernatants from all infected cells were passaged onto fresh cells every days for a total of three passages. viral rna was extracted from cell culture supernatants, and the presence of specific viral rna was evaluated by qrt-pcr as described above. to assess the interferon (ifn) competence of the cells, cells were seeded in -well plates at a density of × cells/ml and grown in dmem containing % fcs and supplements as described above. the following day, cells were either transfected in triplicates with μl of total rna from vsv-infected cells (vsv-rna) using the xtreme gene sirna transfection reagent (roche, basel, switzerland) to stimulate the ifn response of the cells [ ] or cells were left untreated as control. eight hours after transfection, all cells were infected with the ifnsensitive rvfv clone carrying a renilla luciferase [ ] . h after infection, cells were treated with lysis buffer and renilla luciferase activity was measured in a microplate reader. in order to assess the role of cotton rats as an experimental animal model for viral diseases and as a reservoir of zoonotic viruses in the wild, a review of the literature was performed. all studies that described cotton rats as experimental animal models for viral research, and all studies that described an association between viruses (via direct detection by pcr, or viral isolation in cell culture and antibody findings) and wild cotton rats were included ( table ) . outgrowth of primary airway and kidney epithelial cells from cotton rat tissue samples was observed - days after the initiation of the cell culture. outgrowing cells displayed a homogeneous, cobblestone-like morphology typical of epithelial cells in both the airway and renal epithelial cell cultures (fig. ) . successful immortalization was achieved by lentiviral transduction of the large t antigen of sv when the first patches of primary cells were visible in the cell culture dishes. both airway epithelial (subsequently termed shispaec.b) and renal epithelial (subsequently termed shisprec.b) cell lines showed rapid increases in cell growth - weeks after immortalization. to generate a homogeneous cell line, subclones were obtained and further characterized. by endpoint-limiting dilution, single-cell clones were selected and two subclones were used for further experiments, which were subsequently termed shispaec.b- and shisprec.b- . both of these cell lines displayed epithelial cell morphology. the immortalized cell lines and the subclones generated in this study showed expression of pan-cytokeratin and zonula occludens protein, confirming that the cells were of epithelial origin. cytochrome b pcr amplification and sequencing of the product confirmed the host species (data not shown). shispaec.b- and shisprec.b- were tested for their ability to respond to external stimulation of the interferon system. in order to stimulate the ifn response cells were transfected with total rna from vsv-infected cells which was shown to trigger the rig-i and mda dependent ifn signalling cascade [ ] . in comparison to venezuelan equine encephalitis virus [ ] [ ] [ ] eastern equine encephalitis virus [ ] untreated cells (fig. , light column) , vsv-rna transfected cells (fig. , dark columns) showed a -fold (renal cells) to -fold (airway cells) reduced replication of a highly ifn-sensitive rvfv-renilla reporter virus. the pronounced decrease of rvfv-renilla replication reflects the efficient induction of an antiviral state in both cell cultures. overall, these data show that both subclones harbour an intact ifn response to external stimulation with airway cells showing a higher stimulation than renal epithelial cells. shispaec.b- and shisprec.b- were infected with vsv and rvfv with two different mois, and the supernatants were harvested at different time points (fig. ) . vero e cells served as controls and were treated in parallel. both cell lines exhibited a cpe and cell death after vsv and rvfv infection (data not shown). a . log increase in vsv viral rna genome equivalent (ge) copies was seen after infection with an moi of . for the airway epithelial cells, and a . log increase in ge copies was observed for the renal epithelial cells (fig. a) . vero e cells showed an increase in ge copies of almost log with the same experimental set-up. upon infection with an moi of . , the maximum increases in log ge copies were approximately one log lower than those with an moi of . , with the highest ge copy numbers reached in vero e cells ( . log increase), followed by the airway epithelial cells ( . log increase), and the renal epithelial cells ( . log increase). production of infectious vsv particles was assessed h after infection by titration of supernatants on vero e cells, resulting in more than log pfu/ml in all three cell lines after infection with a moi of . and . (fig. b ). upon infection with rvfv at an moi of . , a maximum increase of . log in viral rna ge copies was observed in the airway epithelial cells, and a . log increase in ge copies was seen for the renal epithelial cells. vero e cells showed an increase of . log ge copies. infections with a lower moi of . showed comparable growth kinetics with slightly lower maximum increases in viral rna (fig. c) . production of infectious rvfv particles was assessed h after infection by titration of supernatants on vero e cells. the highest number of plaque-forming units was seen in shis-prec.b- with . log pfu/ml, followed by vero e and shispaec.b- with . log and . pfu/ml after infection with an moi of . . comparable results were observed after infection with a lower moi resulting in . ; . and . log pfu/ml, respectively, for shisprec.b- , vero e and shispaec.b- (fig. d) . to assess s. hispidus cell susceptibility to viruses from the flaviviridae family, infection experiments with wnv strain new york and tbev were performed with two different mois, and the supernatants were collected at different time points. vero e cells served as controls and were treated in parallel. upon infection with tbev, both s. hispidus cell lines and vero e cells showed a cpe and rapid cell death within h (data not shown). the maximum increase in viral rna ge copies was . log for the airway epithelial cells, . log for the renal epithelial cells, and . log for vero e cells after infection with a moi of . . comparable growth kinetics were seen after infection with a moi of . . production of infectious tbev particles was assessed h after infection by titration of supernatants on bhk-j cells. tbev infectious particles were produced by all cell lines in comparable amounts of approximately log pfu/ml after infection with a moi of . and . (fig. b) . for wnv, no increase in viral rna was seen for the airway epithelial cells at either moi. for the renal epithelial cells, a maximum increase of viral rna ge copies of . log was observed, and a . log increase in ge copies was seen for vero e cells. comparable growth curves were seen for the lower moi of . production of infectious wnv particles was assessed h after infection by titration of supernatants on bhk-j cells. the highest number of plaque-forming units was seen in veroe cells with approximately log pfu/ml, followed by lower titers in shis-prec.b- with . and . pfu/ml after infection with an moi of . and . , respectively. no production of infectious particles was seen in shispaec.b- cells (fig. d) . to assess the susceptibility of s. hispidus cell lines to members of the rhabdo-, bunya-, and mesoniviridae families, further infection experiments were performed with recent novel virus isolates from insects [ ] [ ] [ ] . both airway and renal epithelial cells were inoculated with isolates of moussa, ferak, and cavally viruses with a moi of . no cpe was seen with daily observation. supernatants were tested by viral specific qrt-pcr at the end of each passage, which did not reveal an increase in viral rna, thus arguing against replication of these viruses in the cell lines generated in this study (fig. ). in the work presented herein, we generated epithelial cell lines from the respiratory and renal tracts of a cotton rat due to its susceptibility to a broad range of human viruses, as well as the association of multiple important and emerging zoonotic viruses with this species. s. hispidus is a rodent species with a long-standing history as an experimental animal model for virus research. although the first animal experiments on cotton rats date back to the s, only two cell lines from cotton rats are available to date. however, in contrast to experimental animals, cell lines are a less laborious model system, less expensive, and can be used in large-scale viral experiments such as in virus isolation trials without the ethical considerations that are involved in animal experiments. from the cotton rat, an osteoblastic cell line was previously derived from an osteogenic sarcoma (ccrt), of which two lymphoid cell lines (cr-t and cr-t ) were also derived [ ] . however, these cell lines are used for the induction of tumours and as hybridoma cells to produce antibodies. no evaluation of these cells for their use in virus research has been performed despite a large range of viruses that have been investigated in s. hispidus animal experiments. moreover, these cell lines are tumour cells that may not adequately resemble cells in vivo to study virus-host interactions, and they are not derived from target cells that are relevant to the natural course of a viral infection, such as epithelial cells. the value of species-specific cell lines has been shown particularity in the field of bat-borne viruses, where the use of bat cell lines has contributed significantly to studies of novel viruses, virus evolution, and virus adaptation during cell culture as well as replicative capacity and expression of host receptors [ , , , [ ] [ ] [ ] (for a review see [ ] ). cell lines derived from potential reservoir and intermediate hosts can serve as a valuable surrogate to study the replicative capacity of emerging zoonotic viruses, as has been demonstrated for the recently emerged viruses mers-cov and ebola virus by work from our group [ , , , , ] . in the current study, we evaluated the replicative capacity of viruses belonging to the four families bunyaviridae, rhabdoviridae, flaviviridae, and mesoniviridae in s. hispidus epithelial cell lines. several members of the bunyaviridae family were already shown to infect cotton rats, including black creek canal virus (bccv), which belongs to the genus hantavirus. this virus was isolated from the lungs and spleens of cotton rats, and it was further shown by serologic analysis that s. hispidus was the primary rodent reservoir of bccv [ , ] . other hantaviruses associated with s. hispidus are bayou virus and muleshoe virus [ , ] . additionally, from the genus orthobunyavirus, an isolate termed zegla virus was obtained from s. hispidus [ ] . here we showed that s. hispidus epithelial cells are highly susceptible to rvfv, a bunyavirus belonging to the genus phlebovirus, with comparable growth kinetics to interferon-deficient vero e cells. furthermore, we tested the susceptibility of s. hispidus cells to a recently isolated bunyavirus termed ferak virus that belongs to the sister taxon of the genus orthobunyavirus. interestingly, no growth of this virus was seen in the s. hispidus cell lines, suggesting an insectspecific replication cycle for this virus [ ] . the further use of these cell lines for rodent-associated bunyaviruses such as hantaviruses should be evaluated in light of the promising findings for rvfv demonstrated herein. for the rhabdoviridae family, there have been serological findings in cotton rats that suggest a role for this species in the natural cycle of these viruses. specifically, it was shown that neutralizing antibodies to both indiana and new jersey serotypes were found in s. hispidus in a vsv enzootic area in costa rica. antibodies against either one or both serotypes were only found in s. hispidus, and not in exposed mus musculus [ ] . our in vitro results showed that vsv replicates readily in s. hispidus cell lines with high replication titres of up to almost log ge copies, which is approximately only one log lower than that of the replication titres seen in vero e cells. these findings suggest that the s. hispidus cell culture models could serve as suitable in vitro models for further studies on vsv. to further assess the replication capacity of other rhabdoviruses in s. hispidus cells, the insect-derived moussa virus was used [ ] . moussa virus was isolated from mosquitoes that also feed on mammals, but thus far, viral replication in human, hamster, or porcine cells has not been successful [ ] . however, in line with the findings of a study by quan et al., no replication of moussa virus was seen in our experiments with s. hispidus cells. additionally, another insect-derived isolate of a novel virus family termed mesoniviridae was tested on our s. hispidus cell lines. here, no replication of cavally virus on the newly generated cells was seen. a strong association has been reported between the cotton rat and several zoonotic flaviviruses, including wnv, slev, san perlita virus, and cowbone ridge virus [ , , , ] . furthermore, cotton rats have been discussed as potential reservoir hosts in the wild for arboviruses, by which infected viremic cotton rats serve as a reservoir for arthropods that feed on them. in our cell culture experiments with tbev and wnv, we saw replication and production of infectious virus particles in both s. hispidus cell lines for tbev and in the kidney epithelial cells for wnv. moreover, both replication titres were only one log lower than that for vero e cells, indicating a high susceptibility of these cell lines to flaviviruses. additionally, we have used s. hispidus cell lines for the evaluation of a novel sylvatic isolate of slev in an earlier study [ ] . here, it was shown that the endemic strain of slev, termed msi- , replicated in s. hispidus kidney cells. in contrast, the novel sylvatic slev isolate, termed palenque strain, did not show any replication. as s. hispidus has been described as a natural reservoir host for slev, these findings suggest that the sylvatic isolate has not yet adapted to hosts that live outside the primary rain forest, whereas the endemic strain has [ ] . in line with the findings obtained for slev, our results showed that wnv only replicated in kidney cells but not in airway epithelial cells, suggesting that kidney cells are more susceptible to this virus than airway cells. taken together, the multiple in vitro findings presented herein for flaviviruses provide evidence that cotton rats may be reservoirs for multiple members of flaviviridae in the wild. therefore, s. hispidus cell lines, especially s. hispidus kidney epithelial cells, may provide a useful model for in vitro virus-host interaction studies. newly generated epithelial cell lines from s. hispidus are able to support the replication of virus species from important zoonotic virus families, and may therefore serve as valuable tools for studies focusing on the isolation of novel viruses and virus-host interactions. emerging infectious diseases: a -year perspective from the national institute of allergy and infectious diseases the challenge of emerging and reemerging infectious diseases global trends in emerging infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia ecological dynamics of emerging bat virus spillover a novel rhabdovirus isolated from the straw-colored fruit bat eidolon helvum, with signs 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establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice the cotton rat (sigmodon hispidus) as an experimental model for studying viruses in respiratory tract infections. ii. influenza viruses types a and b measles virus replication in lungs of hispid cotton rats after intranasal inoculation replication of clinical measles virus strains in hispid cotton rats use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections use of cotton rats for preclinical evaluation of measles vaccines pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (sigmodon hispidus) distribution of a rodent-adapted strain of poliomyelitis virus in the cotton rat studies of a murine strain of poliomyelitis virus in cotton rats and white mice hiv type- infection of the cotton rat (sigmodon fulviventer and s. hispidus) we thank marco marklewitz, pascal trippner and anne kopp for help with novel arthropod-derived viruses; beate kümmerer and janett wieseler for viral isolates of wnv and tbev; and bettina dubben for organ samples. we thank friedemann weber (university of gießen) for providing rvfv clone and rvfv-renilla reporter virus and alexander pfeifer, (university of bonn) for providing large t antigen lentiviruses. the work was funded by the german research platform for zoonoses and the federal ministry of education and research (grant no. ki to ie). the authors declare that they have no competing interests.authors' contributions ie, le, mam, sj, and cd designed the experiments; le, jt, jp and rk performed experiments and analyses; le and ie wrote the manuscript; and all authors contributed to, read, and approved the final version of the manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -mxyxwkhx authors: sallie, richard title: replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: mxyxwkhx much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. rna polymerases (rna(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. thus, rna(pol )causes more morbidity and premature mortality than any other molecule. the extraordinary genetic heterogeneity defining viral quasispecies results from rna(pol )infidelity causing rapid cumulative genomic rna mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking rnapol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral rna is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. this mechanism – "viral receptor disease (vrd)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type ii diabetes mellitus, and some forms of obesity. viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. many of the world's population suffer from acute and chronic viral infection. the two common types of chronic viral hepatitis (cvh), hepatitis b (hbv) and c (hcv) are major causes of death and morbidity; conservative esti-mates suggest million people are persistently infected with hbv, while hcv may infect a further million. annually, in excess of two million people will die from cirrhosis or liver cancer caused by cvh, and many more suffer chronic ill health as result. during the years since the human immunodeficiency virus (hiv) was identified, perhaps million people have become infected worldwide and each year about a million die from resulting immunodeficiency and consequent opportunistic infections, particularly tuberculosis, and other complications. poor countries bear a disproportionate burden of disease caused by these viruses that further exacerbate poverty through pervasive economic disruption and diversion of limited resources to healthcare and disease control. emerging viral pathogens including west nile virus (wnv), the sars coronavirus, endemic viruses like murray valley, japanese, and other encephalitis viruses, dengue and yellow fever, and seasonal influenza, hepatitis a (hav) and e (hev) cause enormous further morbidity and mortality, while pandemic outbreaks of virulent influenza strains remain a constant threat. together, these viruses probably kill more people every ten days than the boxing day tsunami. rna viral infections, including foot and mouth, bovine viral diarrhea virus (bvdv) and hog cholera virus (hchv), cause similar devastation of animal populations with enormous economic consequences. rna polymerases generate massive genetic variability of rna viruses and retroviruses that circulate within infected hosts as vast populations of closely related, but genetically distinct, molecules known as quasispecies. after translation, this genetic variability causes near-infinite antigenic heterogeneity, facilitating viral evasion of host defences. tuberculosis, malaria and other cellular pathogens also express broad cell-surface antigenic heterogeneity, generated by dna-dependent rna pol . thus, rna polymerases probably cause more morbidity and premature mortality in man, and other animals, and greater economic loss, than any other molecule. despite a depressing global epidemiology that strongly suggests otherwise, the immune system is thought to "control" viruses. what practical meaning does "immune control" have for the individual? there is no argument for hbv, and other viruses, high affinity antibody, generated by prior vaccination or other exposures and directed against neutralizing epitopes, will prevent hbv infection (excepting vaccine escape mutations [ , ] ), in part by blocking viral ligand interaction with cell receptors, or that most patients exposed to hbv develop neutralizing antibodies (hbsab), clear hbsag from serum, and will normalize liver function long term. however, even patients who develop robust immune responses to hbv, defined by high-affinity antihbsab and specific antiviral cytotoxic t cell (ctl) responses, will have both "traces of hbv [ ] ... many years after recovery from acute hepatitis" [ ] and transcriptionally active hbv demonstrable in peripheral blood mononuclear cells (pbmcs) [ ] . furthermore, occult hbv is detected in liver tissue of patients with isolated antihbc (i.e. hbsag/hbsab negative) [ ] and in patients with hbsag-negative hepatocellular carcinoma [ ] suggesting, at least some patients, hbv in may persist irrespective of any immune responses, implying long term latency and low level basal replication may be a survival/reproductive strategy for hbv. for most patients, acute hcv or hiv infection results in life-long viral persistence. although many patients develop immunological responses, including specific antibody and ctl reactivity to various viral antigens, these responses have little discernible impact on either hcv or hiv replication that occurs essentially unchecked at rates estimated between and virions per day [ , ] , indefinitely, while progressive destruction of liver or immune cells proceeds, commonly resulting in cirrhosis or liver cancer (for hcv) or death from immune deficiency (for hiv). evidence that prior hcv infection confers no protective immunity against heterologous hcv infection in humans [ ] or chimpanzees [ ] or against either homotypic [ ] or heterotypic [ ] human reinfection, confirmation that active hcv infection persists long after either apparent spontaneous [ ] or treatmentinduced [ ] viral clearance, or that vaccines causing specific antiviral b and t cell responses fail to protect against infection in animals [ ] , and that antibodies to hcv envelope protein e are only detected in animals with persistent infection [ , ] , further undermines the potency of "immune control" and suggests, at least for patients with hcv, the definition of "control" may need to broadened significantly. based on observations that stronger specific cd /cd immune responses with t-helper (th ) cytokine profiles are found more frequently in patients with self limiting viral infections than those who develop chronic viral carriage [ , ] it is thought ability to mount robust adaptive immune responses predicts viral clearance while failure to do so results in chronic viral carriage [ ] . however, detailed and very painstaking studies, albeit in small numbers of chimpanzees [ ] and patients following antiviral therapy [ ] , have failed to demonstrate any relationship between t cell responses and viral clearance. although development of th and other immune responses are certainly temporally and, probably, causally related to reduced viral replication and viral clearance the assumed direction of causality (immune response -> reduced viral replication), is not proved by the fact those responses develop, post hoc ergo propter hoc, as comforting a conclusion as it may be to reach. the first part of this paper explores the impact of rna pol fidelity on quasispecies behaviour, specifically in mediating immune avoidance during acute hcv infection. we suggest the primary event causing reduction in viral replication is inhibition of rna pol processivity by variant viral proteins, specifically envelope and envelope-related proteins. we also suggest that immune responses to viruses are thwarted initially by broad antigenic diversity generated by low rna pol fidelity but develop, when they do, after viral replication falls (because of reduced rna pol processivity) and polymerase fidelity increases -linked events that occur because of replicative homeostasisthus restricting antigenic diversity sufficiently to permit focused immune recognition. we further suggest immune responses strategically exploit replicative homeostasis to force viruses to reveal critical dominant antigenic epitopes, facilitating progressively more focused immune responses. the second part explores the ineluctable consequence of viral rna quasispecies: that is, translation of rnas into protein quasispecies with a spectrum of phenotypes and unpredictable properties, among which may be disruption of the cell surface receptors that viruses co-opt for cell entry. this innate property of viral quasispecies may explain a wide variety of diseases apart from viral autoimmunity. acute hcv and hbv infection have characteristic kinetics of viral replication, adaptive immune responses, and cause predictable tissue injury, reflected in elevated serum aminotransferases. these kinetic and transaminase responses are summarized schematically for patients with persistent infection (figure ) [ ]. initial hcv replication is very rapid and viral load increases exponentially until about week , at which point viraemia increases more slowly, and asymptotically, towards ~ genome equivalents (geq)/ml by weeks - (these kinetics alone suggesting competitive inhibition of rna pol ). this exponential increase of viral rna in serum reflects explosive dissemination of virus in tissues, detectable by in-situ hybridisation throughout hepatocytes, including the nuclei, within days of infection [ ] . viral replication declines rapidly from weeks - to weeks - falling by - geq/ml but lower level (~ geq/ml) fluctuating replication persists, generally indefinitely, thereafter. by contrast, neither hbv dna nor hbv antigens are detectable in either viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection figure viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection. data summated from figure [ ] and modified to represent typical patients with chronic viral persistence. note: a) high level hcv replication for - weeks prior to any immune responses, b) onset of humoral immune response well after down-regulation of viral replication [ ] , and c) transaminase peaks occurs ~ weeks later. both hbv and hcv are non-cytolytic and viral clearance from hepatocytes, as well as hepatocyte injury, thought to be immune mediated. however, for both hbv and hcv the brisk fall in viral replication following acute infection paradoxical hcv replication kinetics figure paradoxical hcv replication kinetics. if host immune clearance forces (i c , black arrows) reduce viral replication acutely (point a), then they must exceed viral expansive forces (v e , grey arrows) at that point. at equilibrium (e.g. points b through d), viral concentrations (-) and, therefore, viral forces, have fallen by - hence, immune forces i c must fall by > - from a to b for equilibrium to develop. there is no evidence this happens. http://www.virologyj.com/content/ / / precedes the peak of transaminase rise by at least two weeks (figure ). if falling viral replication is due to adaptive immune responses causing hepatocyte lysis the transaminase peak should either precede or be coincident with falling replication. this temporal relationship is also inconsistent with the belief immune factors cause the falling replication seen during acute hcv or hbv, and is analagous to non-cytolytic reductions of viral replication observed for both hbv and lymphocytic choriomeningitis virus (lcv) experimentally, that suggested either [unspecified] antiviral mechanisms are operative [ , ], or that auto-inhibition of rna pol by viral mechanisms (replicative homeostasis) occurs [ ] . however, if other non-cytopathic host anti-viral mechanism(s) are responsible, the kinetic paradox implies their potency falls significantly between points a and b. hepatitis c replication kinetics and their relationship to immune responses are well documented [ , ] but reveal an unexplained paradox. despite high level viral replication, adaptive cellular immune responses to hcv are completely undetectable for at least - weeks [ ] after infection, while humoral responses are rarely detected before - weeks [ ] , and in some patients [ ] , and some chimpanzees [ ] , are never detected at all. an exhaustive and very careful review of the clinical and experimental data relating adaptive immune response and hcv replication kinetics has been published recently [ ] . seeking to rationalize the enigma posed by a complete lack of immune responses to hcv replication of ~ - geq/ml at week but [variable] immune responses to replication at ~ geq/ml after week , the authors conclude "..[the data]...appear[s] to be consistent with the interpretation that hbv and hcv are ignored by the adaptive immune system for about months after primary infection" and "[in hcv].. the adaptive response seems to really ignore for several weeks a substantial quantity of virus (at least copies/ml)..". this is certainly an accurate synthesis of an extensive and highly complex literature but does it make any sense? if adaptive immune responses really ignore high level hcv replication for two months, as suggested, then the following mechanism(s) are implied: a) an accurate mechanism for prompt detection of infection; b) a timing mechanism; c) a trigger mechanism for immune responses independent of any viral factor (given levels of virus are greater before immune recognition than afterwards the trigger for immune response must be either non-viral or falling (!) viraemia); and, as cytomegalovirus (cmv)-specific cd (+) t cell responses arise within days of cmv infection [ ] ; d) a mechanism allowing the immune system to differentiate hcv from cmv and other viruses (and reasons to do so). while possible, this seems unusually inelegant and pointlessly counterproductive, especially as events soon after infection probably determine whether virus is cleared or chronic infection develops. it is much more likely that adaptive cellular or humoral immune responses do not develop in the first - weeks of hcv infection simply because the virus isn't "seen". why should hcv replicating at - geq/ml at week be invisible to the immune system but visible when replicating at geq/ml long term? dissection of this problem requires explicit analysis of what is being measured and how. assay of hcv rna and detection of hcv by immune responses measure two quite different things. quantitation of hcv is typically performed by branch-chain cdna assay (bdna) or quantitative pcr (qpcr) using probes or primers complementary to conserved 'untranslated ( 'utr) hcv rna sequences. immune responses to hcv typically "measures" envelope proteins translated from envelope-encoding rna (eerna) sequences and are directed at specific antigenic amino acid sequences and polypeptide conformations, not total viral envelope protein concentrations. while concentrations of 'utr rna will be proportional to eerna concentrations in any given sample, they may not be identical for two reasons; i) rna transcription may prematurely terminate making 'utr rnas relatively more prevalent than eernas and ii) hcv 'utr is highly conserved, while eerna s are less constrained, making hybridization efficiencies of pcr primers or bdna probes greater for 'utr rnas than for the population of eernas, causing relative under-estimation of true envelope rna concentration . nonetheless, as 'utr hcv rna concentrations will be proportional to eerna concentration, the question remains; why should envelope proteins translated from eerna sequences present at concentrations corresponding to ~ 'utr geq/ml at weeks be visible immunologically, but envelope proteins derived from eerna sequences corresponding to ~ - 'utr geq/ml at - weeks remain unseen? quasispecies biology, specifically variable rna pol fidelity, replicative homeostasis, and sequence-specific requirements for both genetic and immunological detection suggest an answer. rna viruses replicate by copying antigenomic templates, a process catalysed by rna pol , an enzyme lacking fidelity or proof reading function [ ] [ ] [ ] [ ] . theoretically, an rna viral genome like hcv (about bases) could assume any of (about . × ) possible sequence combinations exceeding, by some margin, population estimates of protons in the known universe (about ), meaning the potential complexity of rna viral quasispecies is infinite, for all practical purposes. an rna pol fidelity rate of - errors per base copied predicts at least one and as many as (estimated for hiv) [ ] genomic mutations will be introduced during each cycle of replication. furthermore, as hcv replication results in synthesis of ~ virions per person per day [ ] , on average, mutations will develop at each genomic locus ~ times/day, while the probability any two genomes synthesized consecutively will be identical is about - . the sum effect is inexorable accumulation of genomic mutationsthat, by itself, should threaten replicative fitness because of muller's ratchet [ ] -and progressive dilution of wildtype genomes (figure ), processes that make long-term stability of rna virus quasispecies highly paradoxical [ ] . as argued previously, a combination of selective genomic replication and variable rna pol fidelity, both mediated by replicative homeostasis, act together to prevent rna quasispecies extinction [ ] . the phenotypic consequences of viral quasispecies biology may be more important. progressive divergence of genomic rna sequences away from wild-type sequences caused by rna pol infidelity generates a massive population of closely related, but genetically distinct, rna molecules (figure ), an effect operative at all scales from each open reading frame (orf) to whole virus species. a quasispecies of orf rnas has but one inevitable outcome; translation of a quasispecies of viral proteins with a vast and highly variable spectrum of phenotypes, some subtly nuanced, others grossly defective. furthermore, mutations simplified, two dimensional clade diagram of hyperdimensional viral rna and protein sequence-space figure simplified, two dimensional clade diagram of hyperdimensional viral rna protein sequence-space. because of rna pol (p) infidelity and müller's ratchet, mutations ( ) are introduced into each rna template synthesized, and progressively accumulate, resulting in an rna quasispecies with sequence progressively divergent from consensus sequence. translation results in a spectrum of proteins ( , , , etc.) with properties that also vary progressively from wild-type sequence ( ) to highly variant proteins ( , , etc.). some rnas will be so abnormal that translation or replication fails or is truncated ( ), while others will code for grossly defective proteins ( , etc.). that create new, or obliterate pre-existing, start or stop codons in a significant proportion of rnas, will cause translation of highly unusual and heterogeneous proteins, particularly during high-level viral replication, a phenomenon that may explain hbeag. viral quasispecies cannot, and will not, produce homogeneous proteins with predictable and consistent phenotypic and antigenic properties. while rna pol infidelity will cause progressive divergence of copied sequences away from wild-type or consensus sequences, the probability of any particular sequence arising will fall dramatically with increasing genetic distance from that consensus sequence (figure ), allowing conceptual representation of the resulting genomic (and consequent phenotypic) diversity as a frequency distribution curve, with increasingly variant sequences surrounding a 'centre of gravity of replication', formed by wild-type sequences. viral quasispecies occupy hyperdimensional sequence-spaces, hence any physical representation is necessarily simplified, but because mutation away from wildtype sequences is equally probable in all directions, variant rna and protein frequencies will be normally distributed and the standard deviation (sd, σ) -insofar as 'normal' or 'standard' can be applied to a hyperdimensional space -of that distribution will be a function of rna pol fidelity; if rna pol is completely faithful, the rnas and proteins will be monoclonal and σ = ; if rna pol has no fidelity, rna will be synthesised randomly, and all rna and consequent protein sequences will arise with equally probability, therefore σ = ∞. while viral rna and related protein sequences are theoretically unconstrained (at least before any consideration of functionality), the sequence specificities of any reagents used in their detection (bdna probes, pcr primers, mabs etc) are not, by definition, and their specificity and the efficiency with which they detect variant molecules will fall progressively the further those variant sequences are from the consensus sequence. a zone of 'reagent specificity' may therefore be defined probably encompassing wild type and some variant sequences, but there will exist some rna sequences and corresponding proteins of any quasispecies that are undetectable with these sequence-specific reagents. a threshold of detection of any assay (including immune detection) may similarly be defined; rna or protein sequences present at concentrations below this conceptual level being undetectable by that particular assay. the hcv "early replication" paradox now partially resolves; the 'utr sequences are both highly conserved and common to virtually all rnas in the quasispecies, therefore, the 'utr concentration -that is, the common measure of hcv viraemia -corresponds to the area under the frequency distribution curve. by contrast, envelope rna sequences (and related envelope proteins) are not so constrained and their relevant concentrations (i.e. whether or not that rna or protein sequence is detectable) corresponds to the frequency of that specific sequence in the quasispecies and that, in turn, depends on rna pol fidelity; if rna pol fidelity is low, the frequency or concentration of any particular rna or protein sequence will also be low and may be below the detection threshold, while increasing rna pol fidelity may increase sequence frequency [i.e. the concentration of specific proteins] above detection threshold. but why should specific eerna sequence frequencies -in other words, hcv rna pol fidelity -increase after week , facilitating adaptive immune responses? viral autoregulation, specifically replicative homeostasis, provides an answer. interactions among species, whether between humming birds and flowering plants, primitive viroids and prokaryotic cells or hcv and man, results in an unremitting process of adaptation and responsive counter-adaptation -in effect, a molecular arms race -for each species just to maintain ecological parity. the price of survival for a species is continual evolution. survival, for viruses, requires cell entry, a precondition long antedating necessity to evade more complex host defenses, including interferons and other cytokines and adaptive immune responses, while for cells, and complex cellular organisms, cell wall defenses, including receptor polymorphisms, form a principal barrier against viral invasion. viral survival -effectively meaning rna pol survival -on an evolutionary timescale, as argued previously [ , ] , requires control of mutation and replication rates in a manner adaptively responsive to constantly changing biota and this implies dynamic linkage of rna pol fidelity and processivity with quasispecies phenotypic and antigenic diversity, meaning an autoregulatory linkage -replicative homeostasis -between rna pol fidelity and processivity and envelope proteins, as argued previously [ ] . by definition, evolutionary co-adaptation occurs in response to adaptations in locally prevalent interacting species. natural selection for beak variation(s) in darwin's finches occurs as a consequence of concrete survival benefits these variations -mediating, for example, enhanced food harvesting interactions with other variable plant or animal species -confer to individual galapagos island birds, rather than any inexorable hypothetical 'improvement' in beak function for finches in general. if a species is widely distributed in space, but population mixing is slow or incomplete, locally prevalent interactions with other species will vary and regional genetic variations will arise and be maintained, hence progressive divergence from the original genotype (speciation) may result. for viruses, and their hosts, genetic variations -reflected in viral genotype and cell surface polymorphisms and resulting disease susceptibilities -would be predicted, and are observed [ ] [ ] [ ] [ ] [ ] [ ] , to have frequencies that vary geographically. consider the following; an enzyme (e) functioning in a closed system synthesizes either product a or b that both interact with e to influence output such that a:e interactions cause production of b, while b:e interactions pro-duce a. irrespective of starting conditions (excluding substrate exhaustion and product inhibition), an equilibrium will eventually develop ( figure ) with the relative concentrations of a:b determined by the relative association constants (k) of a:e (k a:e ) and b:e (k a:b ) and the velocity (ν) of production of a from b:e (ν a ) and b from a:e (ν b ). removal or addition of either a or b will alter equilibrium conditions but not the fact equilibrium is reached; if a is removed, for example, the increased two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent spe-cificity (red shading) and threshold of detection (tod) of any assay figure two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent specificity (red shading) and threshold of detection (tod) of any assay. as mutations ( , ) accumulate and rna sequence progressively diverges from consensus sequence ( ) the probability of that rna sequence and corresponding protein (e.g. envelope, env.) arising falls rapidly. standard deviation (σ) of frequency distribution is proportional to rna pol fidelity. frequency genetic distance threshhold of detection (tod) reagent specificity ♦ ♦ frequency of b:e interactions will cause compensatory increased a synthesis; in this sense enzymatic autoregulation occurs. intuitive analysis suggests that enzymes acting in a milieu of increasing concentrations of inhibitory molecules become progressively less processive until reduced enzyme output is insufficient to further inhibit enzyme activity, and an equilibrium state is reached. considering viral replication, if alteration of rna pol fidelity causes synthesis of either wild-type or variant rna sequences (simplified, as a continuum between these two must exist) that are subsequently translated into either wild-type or variant polypeptides that then interact with rna pol such that wild-type: rna pol are high affinity interactions that induce rapid, low fidelity rna pol replication while variant protein: rna pol interactions are low affinity and cause high fidelity rna pol replication at low rate then an equilibrium will eventually develop. hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of rna pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. stable, highly reactive equilibria not only develop as a result of rna pol /envelope interactions and viral autoregulation, there is no option but for this to occur. generation and maintenance of polymorphisms, that is, replacement of existing genes -that, by operational dar-winian definition, have proved their functionality and evolutionary fitness by surviving to reproduce -with variant genes (polymorphisms) of uncertain functionality, fitness or overall compatibility within an organism, is an evolutionary strategy that will only be sustained on a geological timescale if new polymorphisms confer survival benefits to organisms that exceeds the risks and metabolic costs of generating and sustaining those polymorphisms. for primitive cells, lacking functional humoral, cellular or cytokine defense mechanisms, development of cell-surface protein polymorphisms is an obvious adaptive strategy to thwart invasion by primitive viruses. like other adaptive strategies, cell-surface polymorphisms are strongly selected for, and have been highly conserved over deep time, and are found in all organisms from primitive prokaryotic cells [ ] and thermophilic bacteria [ ] through to plants [ ] as well as mammalian cells, strongly suggesting a critical evolutionary function. the lock and key hypothesis, for which there is very considerable evidence [ ] [ ] [ ] [ ] , first proposed by jbs haldane [ ] , contends polymorphisms arise, and are maintained, as protection against cellular parasitism, particularly by viruses . while dna-encoded protein polymorphisms form necessary defenses against viral access, they may not be sufficient; a quasispecies of cells (e.g. the liver) expressing similar and static receptor variations renders those cells vulnerable to sustained attack from any virus that successfully invades any one cell, and further dynamic modification of cell receptors, triggered by viral infection and mediated at the transcriptional level by modulation of dna dependent rna polymerase fidelity in nearby uninfected cells, by a mechanism similar to replicative homeostasis would seem possible. a clear, unambiguous band at the "c" position on a sequencing gel, causes "cytosine" to be assigned to that genetic locus. but does this certitude reflect reality, at least for viral rna quasispecies? direct pcr sequencing is an "averaging" procedure revealing the most frequent nucleotide at any particular locus. however, nucleic acids and proteins cannot express 'an average', and discrete quanta of specific nucleotides or amino acids are present at every locus. a typical clinical serum sample, containing × geq/ml hcv and mutating at - substitutions/base, will contain examples of each possible nucleotide at every locus, but most variations will remain undetected during sequencing or any other method of quasispecies analysis. analysis of cloned dna gives cleaner data than pcr sequencing but if clones (and multiple hcv quasispecies clones are highly unlikely to be identical) provides definitive sequence, would we process the st to reveal different and, potentially, critical sequence variations? and if we did, how would we recognise its importance? is important sequence likely to be present at frequencies of http://www.virologyj.com/content/ / / < %? infectious virions containing, presumably, fulllength functional genome and corresponding wild-type proteins, are often outnumbered by ~ × : in serum by defective and non-infectious particles [ ] that presumably do not, suggesting that important genetic sequence and associated phenotype may occasionally be extremely rare. how the immune system recognizes uncommon, nondescript, but important protein sequences in a featureless background of similar molecules is a non-trivial problem for which replicative homeostasis may suggest a solution. replicative homeostasis, described in detail elsewhere [ , ] , is an epicyclic mechanism of viral autoregulation that results when viral proteins, notably envelope (env), influence rna pol fidelity and processivity. the predicted consequences of replicative homeostasis for rates of intracellular viral replication and mutation, cellular expression of viral proteins and immunological responses occurring because of replicative homeostasis is represented schematically (figures , ). during early viral replication in a naive cell devoid of inhibitory molecules (panel a, a), high affinity wild-type envelope:polymerase interactions predominate, causing rapid low-fidelity polymerase activity resulting in rapid synthesis of variant viral rnas and subsequently proteins, hence causing a broad spectrum of viral proteins to be expressed on the cell surface, each at concentrations below the threshold of immune detection (tod). rna pol infidelity ensures synthesis of variant viral rnas and proteins predominates early, hence variant protein molecules progressively accumulate within cells relative to wild-type viral molecules (panels b-d) and increasing the probability of variant viral envelope:rna pol interactions. variant viral envelope:rna pol interactions causing progressive inhibition of rna polymerase processivity and increasing rna pol fidelity, reducing diversity of viral rnas synthesized and progressively restricting dynamic progression of rna pol functional properties, processivity () and fidelity () predicted by replicative homeostasis figure dynamic progression of rna pol functional properties, processivity ( ) and fidelity ( ) predicted by replicative homeostasis. initial state (a, corresponding to panel a, figure ): in a newly infected cell, high-affinity wild-type:rna pol interactions will predominate resulting in high rna pol processivity but low fidelity causing high-level viraemia with broad virus phenotypic spectrum, maximizing cell tropism. intracellular accumulation of variant viral proteins (b, c.f. panel b, figure ) reduces rna pol processivity but increases fidelity reducing viral rna synthesis and consequently, viraemia before a dynamic, fluctuating equilibrium (c, c.f. panel c or d, figure ) develops in which inhibition of rna pol by variant viral proteins is balanced by increases in rna pol fidelity (with consequent synthesis of wild-type viral products tending to cause high rna pol processivity). conceptual progression of intracellular viral replication events, including variable rna pol fidelity and processivity, restriction of antigenic diversity and immune recognition under influence of replicative homeostasis env viral protein diversity expressed on the cell surface (panels b to d), increasing cell-surface concentrations of individual viral proteins above the threshold of detection (panels c, c) at which point a polyclonal immune response develops. development of low-affinity polyclonal blocking antibodies, restricting cellular egress of viral proteins, further increasing intracellular concentrations of variant envelope proteins, still further increasing the probability of variant viral envelope:rna pol interactions and inexorably further restricting antigenic diversity increasing relative expression of wild-type proteins thus further exposing these epitopes to immune surveillance and facilitating specific high-affinity immune responses, including cytotoxic t cell responses, (d,d) to wild-type proteins. thus, the immune responses can strategically utilize replicative homeostasis to force viruses to reveal important and dominant wild-type epitopes, but those responses develop initially as a consequence of restriction of rna pol fidelity that occur because of replicative homeostasis. high-affinity responses further deplete intracellular concentrations of wild-type proteins, progressively reducing wild-type envelope:rna pol interactions, greatly reducing rna pol processivity to the point of viral latency (e,e), caused by variant viral envelope:rna pol interactions. the hepatitis c "early replication" paradox now resolves completely when considered in the context of replicative homeostasis; initial high level hcv replication (due to high rna pol processivity) remains immunologically undetectable for - weeks, or more, because of low rna pol fidelity causing a broad spectrum of hcv envelope proteins each expressed on cell surfaces at concentrations below the threshold of detection even while viraemia, reflected in concentrations of 'utr rna common to each rna species, are present at - geq/ml. as replication progresses, intracellular accumulation of variant viral proteins increase rna pol fidelity but decrease processivity (replicative homeostasis), downregulating hcv replication and reducing viraemia but restricting antigenic diversity and increasing expression of hcv envelope proteins to beyond the threshold of immune detection. furthermore, the temporal tissue injury (aminotransferase) paradox also resolves in this light: focussed immune recognition (including cytotoxic t cell responses) doesn't develop until after viral antigenic diversity is restricted by replictive homeostasis the transaminase peak would not be expected until after viral replication falls due to autoinhibition of rna pol processivity. varying expression of viral proteins by modulating rna pol fidelity to facilitate immune escape would seem a useful evolutionary adaptation that might be retained by more complex organisms, including cellular pathogens like tuberculosis and malaria, to optimize their stability within hosts. this mechanism of immune avoidance might also explain maternal-foetal tolerance. the human foetus maintains a stable parasitic existence during gestation (and, i expect, to university age and beyond) that is tolerated despite normal maternal immune responsiveness in general and lack of specific tolerance to paternal antigens in particular, a situation made more problematic as expressed foetal antigens are predominantly of paternal origin [ ] . while immunological isolation of foetal tissue by the placental trophoblastic layer [ ] , and placental display of hla-g [ ] , probably contribute to foetal stability in the face of a potentially robust immune attack, neither mechanism would explain persistence of viable foetal nucleated red blood cells within the maternal circulation [ ] in quantities sufficient to permit clinical prenatal diagnosis [ ] . is it possible foetal tolerance is mediated by regulating the fidelity of foetal dna dependent rna transcriptases to ensure any cell-surface antigens are expressed heterogeneously and at levels below the threshold of maternal immune responsiveness? for many classical autoimmune disorders, including primary biliary cirrhosis [ ] , multiple sclerosis, and rheumatoid arthritis, convincing epidemiological evidence [ ] , including cases clustering [ , ] , strongly suggests these diseases are triggered by infectious agents in genetically predisposed individuals. in others, such as diabetes mellitus, tantalizing epidemiological [ ] , clinical [ ] and laboratory [ ] evidence has implicated enteroviruses, but has suggested viral-triggered autoimmune processes, rather than cytolytic destruction of pancreatic betacells [ ] . similar circumstantial evidence exists for myocarditis, demyelinating diseases, myositis and other post infectious inflammatory disorders. when macfarlane burnet wrote autoimmunity arises from "inability to distinguish self from non-self" hbv, hcv, hiv and other viruses, now established to cause diseases with clear autoimmune features were unknown. viral infections, particularly hepatitis c -and its treatment with interferon -are associated with many varied autoimmune phenomena [ ] , and thyroid disease [ ] [ ] [ ] , diabetes mellitus [ , ] , membranous, membranoproliferative and cryoglobulinemic glomerulonephritis, vasculitis and peripheral neuropathy [ ] , and autoimmune gastritis [ ] are all very well documented, although the mechanism(s) are unknown and causality is certain. classical serological markers of autoimmunity, including rheumatoid factor, antinuclear antibodies (ana), anticardiolipin, antithyroid, anti-liver/kidney/microsomal antibodies (anti-lkm), as well as hcv/anti-hcv immune complex formation and mixed essential cryoglobulinemia are common accompaniments of chronic hcv infection [ ] , raising the obvious question of whether all "autoimmunity" has a viral basis. indeed, zinkernagel's pragmatic and subtly anticipatory; "if we know the infection, we call the disease immunopathologically mediated; if we do not recognize or know it, we call the disease autoimmune [ ] " fully reflects recent explosive growth of information and the deeper questions this information poses. rna virus quasispecies biology, specifically the generation of rna quasispecies by rna pol , and translation of these immensely variable rnas into protein quasispecies, suggests an immediate solution to the problem of viral autoimmunity and, by extension, to autoimmunity in general, as well as suggesting a unifying hypothesis to explain other diseases known to have multi-factorial aetiologies that include inflammatory components -such as coronary artery disease -in addition to other diseasesincluding schizophrenia and some forms of depressionthat currently lack rational and coherent pathogenic explanations. viruses are known to co-opt cell surface molecules, including lectins, hormone receptors and cell signaling molecules, to access cells. receptors, and other cell surface molecules, identified as "viral receptors"or to specifically interact with viral proteins include prostaglandins, catecholamines and acetylcholine receptors [ ] , serotonergic neurotransmitters ( ht) [ ] , endothelial cell glycoproteins [ ] , insulin-like growth factor (igf-ir) and its major signaling molecules insulin receptor substrates irs- [ ] and irs- [ ] , epidermal growth factor (egf) [ ] , neurotrophin receptor [ ] , thyroid hormone receptor tralpha [ ] , an immunoglobulin protein superfamily [ ] , low density lipoprotein (ldl) receptors [ , ] , transferrin receptor (tfr) [ ] , asialoglycoprotein receptor (asgp-r) [ , ] , and angiotensin-converting enzyme [ ] , to cite biologically diverse examples. of necessity, some receptor affinity studies have used cloned viral protein ligands, an artificial situation that cannot approach the phenotypic complexity of rna viral protein quasispecies. nonetheless, variable virus receptor affinities [ , ] , evolutionary adaptation of receptor affinity [ ] , emergence of escape variants with altered receptor affinities [ ] , temporal alteration of receptor usage [ ] and capacity to exploit alternative entry pathways [ ] have all been confirmed, suggesting viruses are capable of generating highly plastic ligands with very broad receptor affinities. if a virus co-opts a receptor for cell entry, then wild-type envelope (consensus sequence) epitopes, coded for by wild-type rna sequences, will probably form the common viral ligand. however, any viral rna quasispecies also contain a vast spectrum of rnas derived from, and similar to, envelope open reading frame (orf) consensus sequence, but variant from it. as the envelope orf quasis-pecies sequences progressively diverge from wild-type, the quasispecies of envelope proteins translated from these variant orfs will also, and inexorably, diverge in sequence, structure and biological function from wildtype envelope sequence proteins. some of these envelope proteins will be functionally identical, but others, and probably the vast majority, will range from subtly different to grossly abnormal, either due to major differences of sequence and/or chemical or steric amino acid incompatibility, or because of premature introduction of stop codons. even minor amino acid differences, as sickle cell anaemia illustrates, and has been confirmed specifically for viral receptor usage [ , ] , may catastrophically alter a proteins' function with respect to co-opted viral receptors, with some having no binding affinity, while others will bind strongly and act as agonists, antagonists or competitive inhibitors of normal receptor function. variant and defective viruses, and their polypeptides, will be in vast molar excess compared to wild-type [ ] but will exhibit similarly high antigenic variability, permitting escape from immune and other scavenger mechanisms. as many variant viral polypeptides will bind tightly to "self" receptors, but contain immunogenic non-self motifs, a polymorphic (because variant viral proteins will themselves be highly polymorphic due to the quasispecies process) immune response, apparently directed against "self" antigens, but actually targeting virus protein-receptor complexes virtually indistinguishable from normal cell receptors, will result causing apparent 'autoimmune' tissue damage. this mechanism suggests an explanation for common autoimmune phenomena. if a virus enters cells because wild-type envelope motifs interact with insulin, insulin receptor substrate [ , ] , tsh or related molecules [ ] , or acetylcholine [ ] receptors, many variant envelope polypeptides, generated by envelope orf quasispecies rnas, would have similar receptor binding affinity, but may effectively disrupt receptor function, predictably causing impaired glucose tolerance or diabetes mellitus, thyroid dysfunction, or myasthenia gravis with secondary resistance to, and elevation of, the normal hormone ligand (insulin, tsh etc.). the expected consequences disruption of receptor function by variant viral proteins might explain many common biochemical pathologies; for example, what effect would chronic blockade of parathyroid (pth) receptors by viral proteins have on pth levels, the parathyroid glands, or bone? leptin is a kda protein hormone secreted by adipocytes and carried across the blood-brain barrier by a rate-limiting transporter to act on hypothalamic receptors [ ] where, among other functions, it regulates thyrotropinreleasing hormone (trh) genes and upregulates alphamelanocyte-stimulating hormone and other anorexigenic neuropeptides [ ] important to appetite-regulation and energy balance [ ] . leptin also regulates a broad spectrum of other processes and behaviours including thermogenesis, blood pressure and immune function. s=serum leptin concentrations and leptin resistance, are independent markers of obesity, weight gain, systemic hypertension [ ] , diabetes mellitus [ ] , obstructive sleep apnoea [ ] and myocardial infarction [ ] , while polymorphisms of the leptin gene are associated with insulin resistance [ ] and long-term risk of developing diabetes mellitus [ ] . predictably, variant envelope proteins generated by envelope orf rna quasispecies from viruses utilizing leptin receptors for cell access would have similar receptor affinity, but exhibit non-physiological leptin antagonist or agonist properties, thus disrupting leptin receptor function, altering energy regulation, and causing either excess caloric intake unrestrained by satiety responses, or inappropriate satiety signals with pathologically reduced caloric intake. as clear evidence exists for viral disruption of leptin function [ ] and virus-associated weight gain in humans [ ] and monkeys [ ] , is it possible the global epidemics of type ii diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function? could it also be that ethnically based epidemics of obesity, diabetes mellitus, hypertension and reno-vascular disease (the 'metabolic syndrome'), as seen in pima indians, nauruans and australian aborigines [ ] have developed not primarily because of exposure to "western" foods and lifestyles -that, after all, are all-pervasive without necessarily having so dramatic an effect on other groups -but because of chronic or recurrent exposure to viruses, or genotypes of viruses to which their particular repertoire of receptor polymorphisms confer no protection? or that anorexia nervosa develops, in some patients, when variant viral proteins with aberrant leptin-agonist function arise during the course of viral infection, as the temporal relationship between infection and disease onset, very clearly documented in one study [ ] , suggests. cardiovascular disease, the leading cause of premature death and disability in most western countries, has a wellestablished multi-factorial basis involving a complex interplay between genetic predisposition, environmental and personal risk factors -including systemic hypertension, diabetes mellitus, hyperlipidaemia, obesity and cigarette smoking -and more recently recognized mechanisms, including endothelial dysfunction [ ] , vascular inflammation [ ] and leptin levels [ ] . systemic hypertension, diabetes mellitus and hyperlipidaemia have long-established, but complex, patterns of inheritance, a situation further compounded by evidence receptor polymorphisms -including those of angiotensin ii type receptor [ ] , irs- gene [ ] and low density lipoprotein receptor (ldlr) [ ] -both confer disease susceptibility and have regionally variable prevalences [ , ] . the flaviviradae -including hcv -as a family, and the rous sarcoma virus, utilize low density lipoprotein receptors to enter cells [ , ] , while angiotensin ii [ ] , insulin receptor substrates (irs and irs ) [ ] , and endothelial cell glycoproteins [ ] and other receptors widely distributed in vascular tissues are known to be permissive for virus cell entry establishing, in principle and in fact, viral-protein receptor affinity relevant to cardiovascular diseases. viruses accessing cells through these receptors will generate a quasispecies of variant proteins capable of disrupting receptor function potentially causing hyperlipidaemia, hypertension, hyperglycaemia and endothelial dysfunction, as well as immune-mediated endothelial cell damage, thus establishing the necessary and sufficient conditions and a chain of events that potentially link viruses and vascular diseases, including myocardial infarction. this hypothesis exists at the confluence of established risk factors for coronary artery disease, including genetic susceptibility, polymorphisms predisposing to hypertension [ ] [ ] [ ] , diabetes [ ] and hypercholesterolaemia and substantial new data implicating vascular inflammation [ , ] , endothelial dysfunction [ , ] , leptin dysregulation [ ] and viral infection [ , ] in the pathogenesis of vascular disease. furthermore, this final common pathway can account for that small, but significant, group of patients with vascular diseases but no clinically identifiable risk factors, as well as the non-random co-incidence of depression and coronary artery disease [ ] (as discussed below) in addition to the anti-inflammatory action of hmg-coa reductase inhibitors (statins) [ ] , and their effect in lowering cardiovascular mortality independent of cholesterol reduction [ ] ; if statins compete with variant viral proteins for hmg-coa reductase receptor binding, and displace immunologically attractive molecules, inflammatory responses directed at viral product, but involving endothelial cell receptors, will be ameliorated (figure ). human immunodeficiency virus hiv-associated dementia (hivd) occurs in % of hiv-infected adult patients, and as a major cause of dementia in the young represents "proof of principle" of virus-caused dementia, raising the possibility other forms of virus related dementia exist. although highly active antiretroviral therapy (haart) has reduced the incidence of hiv-d by - % [ ] , it remains a major cause of morbidity and the pathogenesis poorly understood. direct cytopathic effects of hiv or other viruses are unlikely, while active replication of virus, high-level viral protein expression [ ] , and increased viral envelope sequence-diversity in blood and brain [ ] are all important, clearly indicating viral proteins are pathogenically important. the clinical features of hivd, including psychomotor slowing, apathy, and altered gait and posture, strongly suggest a subcortical dementia with involvement of the basal ganglia and striatal dopamine receptor pathways. schizophrenia, depression and bipolar affective disorder, and anorexia nervosa are highly prevalent, chronic conditions of unknown aetiology that cause enormous morbidity and generate significant health care costs. each of these disorders have well documented, albeit regionally variable, associations with receptor -including dopamine -polymorphisms [ , [ ] [ ] [ ] [ ] [ ] , as well as epidemiological evidence that viral infections are aetiologically important, either directly or as precipitating events [ , [ ] [ ] [ ] [ ] , although other sero-epidemiological studies [ ] and work directly seeking viral nucleic acids in patients with schizophrenia have proved negative [ ] . if a virus, or viruses, use dopamine, acetylcholine [ ] , neurotrophin [ ] , serotonergic ( -ht) [ ] , or other neuro-transmitter receptors to access cells (and, given rna virus quasispecies biology, it would be surprising if some didn't), then the rna quasispecies will generate a quasispecies of variant polypeptides potentially reactive to these receptors. while it is difficult to imagine what effect perfusing a functional human brain with a solution of antigenic, inflammatory polypeptides that bind to, and are variably disruptive of, critical neurotransmitter receptor function, might have on cognition, perception, behaviour, attention span, abstract thought, fine motor or emotional control, it is unlikely to be beneficial. in this context, the welldocumented cognitive abnormalities -unrelated to depression -found in patients with early hcv and hiv infection [ ] [ ] [ ] are unsurprising. virus receptor disease (vrd) is quite distinct from either immune complex deposition disease due to deposition of macromolecules in tight vascular arcades, or from disease related to altered cell tropisms and is also completely independent of the primary site of viral replication; both non-inflammatory receptor blockade and immune-mediated inflammation directed at viral protein-receptor complexes could cause pathology of tissues non-permissive for and remote from the primary site(s) of viral replication with "autoimmune" damage to the liver, pancreas, brain, skin or lungs arising, for example, from chronic small intestinal virus infection. viral quasispecies biology predicts vrd will have other characteristics. first, due to replicative homeostasis, the ratio of wild type to variant viral proteins of the quasispecies will both fluctuate with time and will alter dramatically after initial infection; if wildtype proteins are dominantly agonist in function with respect to their receptor, variant proteins, most likely, will predominantly exhibit antagonist function (and vice versa). furthermore, the net effect of viral proteins (because of viral autoregulation) will fluctuate initially between receptor agonist and antagonist function, before becoming predominantly antagonistic, thus providing a possible explanation for transient thyrotoxicosis during early thyroiditis (before hypothyroidism supervenes), for hypoglycaemia seen during early insulin-receptor antibody-mediated insulin resistance [ ] , and for the contradictory functions ascribed to hiv nef [ ] . a corollary of fluctuating phenotypic dominance of viral protein quasispecies is that receptor affinity of these proteins will also fluctuate, and any resulting inflammation may vary in both intensity and anatomical distribution over time. second, because viruses utilize alternate receptors for cell access, apparently homogeneous disease processes could result from multiple different viruses. similarly, because cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship ( ; unbound, ; activated), receptor permis-sive for virus cell entry ( ) or blocked by polymorphism (rp, ) figure cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship ( ; unbound, ; activated), receptor permissive for virus cell entry ( ) or blocked by polymorphism (rp, ). receptor blockade by variant viral envelope proteins (green e, ), blockade by antigenic envelope proteins stimulating "autoimmune"response apparently directed against self receptors (e, ), competitive displacement of antigenic proteins by drug (d, e.g. statin, aspirin) abrogating immune response ( ). virus quasispecies produce a broad spectrum of protein phenotypes, and the receptor polymorphisms permissive for cell entry for specific viruses will be variably distributed in host populations, pathology of widely variable tissues in different individuals could result from the same virus. third, as evolutionary co-adaptation results in progressive genetic co-divergence of interacting species, the receptor polymorphisms predisposing to (or protecting against) infection by any particular virus, and resulting vrd, and the common viruses causing them, would be predicted to vary geographically, an expectation multiply confirmed for disease associated polymorphisms. as a corollary this suggests individuals migrating from regions where hosts and virus strains are stably co-adaptated to other areas, where different viruses are prevalent, might experience increased rates of vrd -beaks optimally adapted for finch survival on the galapagos may be a liability elsewhere -a prediction again amply confirmed [ ] [ ] [ ] ;. finally, if immune mechanisms are unable to clear rna viruses like hcv and do not cause the reduced viral replication seen during acute infection, are they any more likely to be effective against other rna viruses? is it possible that self-limiting infections like influenza and sars also autoregulate their replication, and, like hcv or hbv, become partially dormant, yet remain transcriptionally active, in the face of an active and powerful immune response? pcr amplification of influenza rna from convalescent samples makes this readily testable, while the documented relationship of influenza to myocardial infarction [ ] and juvenile rheumatoid arthritis [ ] makes the question important. if confirmed, the well-documented seasonality of some depressive illnesses [ ] and schizophrenia, [ ] and increased rates of schizophrenia during influenza epidemics [ ] , and the increased incidence of both depression [ ] and schizophrenia [ , ] following in-utero exposure to influenza may be more rationally explained. if quantitative pcr (qpcr) assays of both 'utr and envelope rnas are performed serially, and data expressed as [ ' utr rna]/[env rna] for each sample, then a numerical expression describing changing quasispecies complexity over time may be obtained. in case prescient genius is unappreciated, haldane formulated the "lock and key" hypothesis on the basis of protein polymorphisms, defined by gel electrophoresis, and some general musing about predation and evolutionary struggle, two decades before the nature of dna was elucidated. i have no pecuniary interests, whatever, in this work and do not stand to gain 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and cardiovascular risk migration as a risk factor for schizophrenia: a danish population-based cohort study impact of migration on coronary heart disease risk factors: comparison of gujaratis in britain and their contemporaries in villages of origin in india variability in the -ht( a) receptor gene is associated with seasonal pattern in major depression i thank my wife sophie j coleman, and sons matt and tim, for everything important, my parents dick and janet for extraordinary opportunity, and some great physician-teachers -that most noble vocation -of the university of western australia medical school; professors mike mccall, dick joske, bill reed, bill musk, peter pullan, michael quinlan, dick lefroy and ted haywood. special thanks to karl ruckriegel for turning back-of-envelope sketches into first-class graphics. any remaining lack of clarity is my fault. key: cord- -de lmzl authors: hu, han; guo, nan; chen, shuhua; guo, xiaozhen; liu, xiaoli; ye, shiyi; chai, qingqing; wang, yang; liu, binlei; he, qigai title: antiviral activity of piscidin against pseudorabies virus both in vitro and in vivo date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: de lmzl background: swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. novel antiviral agents need to be developed to control this situation. methods: in this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (amps) against several important swine-origin pathogenic viruses by tcid( ) assay. plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. protection effect of piscidin against pseudorabies virus (prv) was also examined in mouse model. results: piscidin (piscidin ), caerin (caerin . ) and maculatin (maculatin . ) could inhibit prv by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from prv-induced apoptosis. among the peptides tested, piscidin showed the strongest activity against prv. moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with prv. conclusion: in vitro and in vivo experiments indicate that piscidin has antiviral activity against prv. swine-origin virus infection is one bottleneck for the development of the porcine industry worldwide. the pathogenic viruses isolated from pigs including pseudorabies virus (prv), porcine reproductive and respiratory syndrome virus (prrsv), porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and rotavirus (rv) are commonly observed in china. the viral infection is also responsible for the secondary infection by bacteria which has greatly increased the application of antibiotics [ , ] . in the past decades, the efforts to alleviate pig viral diseases have focused on the development of vaccines to enhance the adaptive immunity of the hosts [ , ] . however, some of the evolving viruses could escape from the host immunity through different kinds of strategies. several viral diseases have broken out in recent years among pig herds, such as the reemergence of swine-origin influenza in [ ] , ped in late [ ] and pseudorabies in [ ] . thus, there is an urgent need to develop novel potential agents to kill these viruses or block their infection. amps are common host defense molecules in nearly all forms of life. ever since their discovery, amps have gained worldwide attention as important alternatives in the field of disease prevention and immune modulation [ , ] . previous studies mostly focused on the protection effect of amps against bacterial and fungal infection [ ] . later on, amps were also reported to be effective against viral infection [ ] . it has been reported that some alpha-helical peptides can act as virucidal agents. for example, caerins and maculatins isolated from amphibian skin could completely inhibit human immunodeficiency virus type (hiv- ) infection after virus is exposed to peptides within minutes [ ] . piscidins, discovered in the mast cells of fish, could reduce the infectivity of several important fish-origin viruses [ ] . indolicidin, a natural -amino acid antimicrobial peptide isolated from bovine neutrophils, could directly kill hiv- [ ] . bovine lactoferricin derived from bovine lactoferrin has been reported to exert antiviral activity to fight against human cytomegalovirus (hcmv), herpes simplex virus type (hsv- ), hsv- , and adenovirus [ ] . other antimicrobial agents, such as human defensins which is a group of beta-sheet peptides, can inhibit enveloped viruses such as hsv- and hsv- , hiv- , vesicular stomatitis virus (vsv), influenza virus, and hcmv by directly inactivating viruses [ ] . therefore, amps might be promising agents against viral infections. prv, a large enveloped dna virus, is a swine neurotropic herpesvirus [ ] . although the pigs are the natural reservoir for the virus, most mammals and some avian species are also susceptible to prv. prv infected animals may die from central nervous system disorders [ ] . prv infection poses a severe threat to pig industry and either attenuated live or inactivated vaccines are usually used to control the disease [ ] . although vaccination can suppress development of the disease, vaccines cannot eliminate virus infection. mutant isolates emerged and caused pseudorabies outbreak in [ ] . thus, novel antiviral agents should be developed as a complementary to vaccination. in one of our recently published papers, we described the antiviral activity of caerin against porcine epidemic diarrhea virus (pedv) in vitro [ ] . in this study, we investigated the activity of five amps including piscidin, caerin, maculatin, lactoferricin b, and indolicidin against several porcine-origin viruses. more inhibitory activity of piscidin, caerin, and maculatin against prv was also explored. the in vivo protection effect of piscidin against prv infection was further investigated. chen et al found that piscidin has the anti-inflammatory and anti-nociceptive properties in inflammatory animal models [ ] . kumar et al reported the obvious anti-endotoxin and anti-bacteria properties of piscidin- analogues both in vitro and in vivo [ ] . the peptides (table ) used in this study were synthesized on an automated solid-phase peptide synthesizer by neweast biosiences inc. (wuhan, china) [ ] . the crude peptides were purified via a reverse-phase highpressure liquid chromatography (rp-hplc) using a c column (waters xbridge). the elution was conducted using a water-acetonitrile linear gradient ( - % of acetonitrile) containing . % (v/v) trifluoroacetic aicd (tfa). finally, the purity and accurate masses of the product peptides were determined using hplc and mass spectrometry, respectively. the prv strains of ea, hnxx, , and tgev wh- were propagated in pk- cells based on the method reported in previous studies [ ] . rv tm-a was propagated in rhesus monkey kidney cell line (ma ) [ ] . prrsv ya was cultured in marc- cells [ ] . pedv strain ch/ynkm- / was cultured in vero cells as described before [ ] . the virus suspension was stored at − °c until used. in our initial topical screening assays, the viruses were pre-incubated with peptides ( μg/ml) for h at °c before they were added to the target cells. peptide-free controls (virus plus cell culture medium only) were incubated in parallel. after incubation, the tcid of peptidetreated viruses and peptide-free viruses was measured. residual virus was calculated according to the following formula, residual infectivity = b/a × % ("a" represent tcid of residual virus non-treated by the amps (control), "b" represent tcid of residual virus after treated by amps). a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) cell proliferation assay was used to assess cell viability as described previously [ ] . briefly, pk- cells were added to -well tissue-culture plates (coster, usa) and incubated overnight at °c. serial two fold dilutions of amps ranging from to . μg/ml were added into the plate. the cells were incubated with amps at °c for h, and the morphology of the cells was evaluated under light microscope. then, the cells were further incubated with mtt solution for h and the cell pellets were collected for measurement of absorbance at nm by an elisa reader. the antiviral activity was evaluated by plaque reduction assay [ ] at different infection stages: pre-infection and post-infection, as well as direct inactivation of the viruses. briefly, the direct effect of peptides on virus per se was analyzed by the following procedures. monolayer of to analyze the post-infection effect of the peptides on virus growth, cells were infected with the same amount of virus mentioned above at °c for h. subsequently, the cells were washed times with dmem and treated by the peptides at different concentrations for h at °c. the peptides were removed from the cell culture and replaced with the overlaid medium. the resulting pfu titer was determined as described above. to understand the pre-infection effect of the peptide on virus, the cells in -well tissue culture plates were treated with peptides at serial concentrations at °c for h. then, cells were washed with dmem for times, followed by infection with viruses. the cells were covered with the overlaid medium for plaque assays. cell apoptosis was analyzed with annexin v-fitc kit (nanjing keygen biotech. co., ltd). briefly, fitc-conjugated annexin v ( μl/well) and propidium iodide (pi, μl/well) were added to the cells infected by amptreated viruses. then, the obtained mixture was incubated at room temperature for min in the dark prior to fluorescence observation. cell apoptosis was determined on basis of the observation soon after initiating apoptosis. cells translocated the membrane phosphatidylserine (ps) from the inner surface of the plasma membrane to the cell surface. thereafter, ps can be easily detected by staining with a fluorescent conjugate of annexin v (green), a protein that has a high affinity to ps. cellular late apoptosis was determined by cell staining with pi (red). the results were analyzed by fluorescence microscope (nikon, japan) at hpi. in vivo prv challenge assay the to week-old specific-pathogen-free balb/c mice were purchased from the experimental animal center of zhongnan hospital of wuhan university (china) and randomly divided into six groups consisting of mice each. individuals in each group of mice were anesthetized with - % isoflurane gas and challenged by the intra-footpad injection with μl of dmem containing × tcid of prv-ea in the presence or absence of , , , . μg/ml piscidin. after days, the surviving mice were challenged with × tcid of prv again. mice viability and behaviors were monitored on a daily basis. in the first days after the challenge, the mice commonly did not exhibit severe symptoms. however, mice gradually showed neurological symptoms in the subsequent days during the monitoring period and the post-challenged mice were monitored since day post prv infection once every - h for days to obtain the survival information of the mice. the level of prv infection symptoms was scored for every mouse by the following system: = posture normal, absence of neurological symptoms, = mild neurological symptoms: excitation, unrest, occasional itching, and foot swollen; = severe neurological symptoms: ataxia, severe pruritus, and self-mutilation, biting and bleeding of the footpad. mice were euthanized in the chamber with co gradually filled when the score reached . on day , three mice from control group and noncontrol groups were euthanized and brain tissues were collected and transferred to % paraformaldehyde. the tissues were dehydrated in ascending grades of ethyl alcohol i.e. , and % ethanol. afterwards, the tissues were cleared with xylene. slides were stained with haematoxylin and eosin (h&e) stain and analyzed through a light microscope. all of the animal experimental protocols were approved by the ethics committee of huazhong agricultural university according to hubei province laboratory animal management regulations (hzaumo - ). during the experiments, mice were offered ad libitum access to water and food in a controlled environment of a h light/dark cycle. all efforts have been made to reduce suffering of animals. the antiviral effects of the peptides (maculatin, caerin, piscidin, lactoferricin b, indolicidin) were investigated in vitro against several viral pathogens that severely threaten the porcine industry. four enveloped viruses were used in this assay including the prv ea strain, pedv ynkm strain, tgev wh- strain, and prrsv ya isolate, as well as one non-enveloped rv tm-a strain. the peptides exhibited different inhibitory activities against these viruses (fig. ) . the relative potency of the peptides against prv was: piscidin≈caerin>ma-cualtin>indolicidin> lactoferricin b. piscidin and caerin showed strong virucidal activity with the residual infectivity being around . % while indolicidin and lactoferricin b exhibited mild antiviral activity. in descending order of potency, the peptides against pedv ranked as follows: caerin>piscidin> maculatin>lactoferricin b > indolicidin. caerin had the most potent activity with the residual infectivity being . %. the assay results indicated, that the order of potency versus prrsv was: caerin>lactoferricin b > piscidin>maculatin>indolicidin. caerin exhibited the best inhibitory activity with the residual infectivity being . %, separately. the assay results of antivirus activity against tgev indicated the activity order as follows: piscidin>lactoferricin b > indolicidin>maculatin>caerin. piscidin was the most potent peptide versus tgev with the residual infectivity being . %. the assay result also indicated that prrsv and tgev were less sensitive to the tested peptides, compared with other viruses (fig. ) . when it comes to rv, the relative potency order was: piscidin>caerin>maculatin>lactoferricin b > indolicidin. piscidin displayed the most potent activity against rv with residual infectivity being . %. piscidin and caerin exhibited activity against almost all the viruses with the residual infectivity being lower than % with exception of piscidin versus prrsv and caerin versus tgev. caerin showed the greatest inhibitory activity against pedv with the residual infectivity being . %, which was the lowest value among all the viruses tested. maculatin exhibited strong activity against prv ( . %) and mild activity against rv ( . %). however, it showed limited activity against pedv, prrsv, and tgev. lactoferricin b and indolicidin were not as potent as other peptides to fight against viruses. lactoferricin b showed stronger inhibitory activity against tgev than against other tested viruses with residual infectivity being . %, and residual infectivity of indolicidin versus prv was . %. we examined the effects of amps on cell viability after incubating pk- cells with different concentrations of each peptide. cytotoxicity was evaluated using mtt method (fig. ) . the non-linear regression analysis result indicated that the % toxic concentration (tc ) of caerin, maculatin, piscidin, and indolicidin were . , . , . , and . μg/ml, respectively. indolicidin was fig. spectrum of antiviral activity of the peptides (tcid assay). peptides ( μg/ml) and viruses were incubated at °c for h before they were added to the target cell monolayers. after incubation for - h, tcid of the virus was recorded. the bars represent ± se. three separate assays were conducted (n = ). the dashed line means % residual infectivity fig. cytotoxic properties of the peptides. pk- cell monolayers were incubated with peptides. the cytotoxicity was measured by mtt assay (n = ). the cell survival rates at different peptide concentrations were plotted and the dashed line means % cell survival less cytotoxic than piscidin, caerin and maculatin. lactoferricin b exhibited the weakest cytotoxic activity and it did not exhibit obvious cytotoxic activity even at maximum concentration of μg/ml. to determine whether the inhibitory activity against prv by maculatin, caerin, piscidin was strain-specific or not, we tested the activity of these peptides against different prv isolates including prv-hnxx (a newly prv isolate in china in ) and prv- (modified bartha strain). we found that all the three peptides displayed inhibitory activities against all the tested strains (prv hnxx, prv , and prv-ea) (fig. a) . this finding indicated that the activity of the peptides versus prv was not strain-specific. when the concentration of the peptides increased from to μg/ml, the residual infectivity of prv decreased (fig. b) . this trend was the most obvious for maculatin with the residual infectivity decreasing from . to . %, while the groups treated with piscidin and caerin exhibited a decrease by more than folds in the residual infectivity. these data indicated that these peptides inhibited the virus in a dose-dependent manner. in order to better understand the inhibitory effect of amps on the propagation of prv-ea, we examined a b fig. prv inhibitory activity displayed by maculatin, piscidin and caerin (tcid assay). a prv of ea, hnxx strain and isolate were treated with the peptides ( μg/ml) for h before they were added to the cell monolayers. the bars represent means ± standard errors of the means of three separate experiments (n = ). *, statistically significant difference by one-way anova with tukey post hoc test (p < . ), ns means not significant. b prv ea was treated with peptides ( μg/ml) for h before added to the cell monolayers. the effects of the peptides at μg/ml and at μg/ml against prv ea were compared whether amps directly damaged virus particles or indirectly interacted with the host cells pre-or post-infection. plaque-reduction assay was also performed to confirm the peptides' antiviral activity. the viruses were incubated with the peptides prior to infection. the result showed that all the three peptides inhibited the virus infection in a dose-dependent manner (fig. ) . at the concentration of μg/ml, pisicidin, caerin and maculatin were found to inhibit most of the prv particles. pisicidin exhibited the strongest activity and obvious inhibitory ability at concentrations above μg/ml. the % effective concentration (ec ) values of the peptides were calculated using probit analysis by ibm spss (version , new york, usa). the analysis revealed that ec values of caerin and maculatin were . μg/ml and . μg/ml, respectively. ec of piscidin was the lowest ( . μg/ml) among the peptides tested, which indicated that piscidin was the most potent antiviral peptide against prv in this study. subsequently, the experiments were conducted to further determine whether the peptides could function at post-(curative) or pre-infection (prophylactic) stage. the cell monolayers were treated with piscidin, caerin, and maculatin before (prophylactic) or after (curative) the virus binding stage, separately. no inhibitory effect of the three peptides on prv could be detected (the obtained data not shown). this implied that these peptides probably inhibited prv infection by directly interacting with the virus particles. the cell apoptosis assay showed that prv could induce both early and late apoptosis of pk- cells (fig. ) . the late apoptosis rate of cells infected by the peptides-treated viruses decreased as the concentration of the peptide increased (fig. b) . the inhibition of late cellular apoptosis was obvious at the concentration of μg/ml (fig. a) . in the control group of the in vivo assay, mice (n = ) were injected with μl of piscidin ( μg/ml) by the intra-footpad route. all mice survived and behaved normally. prv was injected into mice with or without piscidin at various dosages ( . , . , , μg/ml). and the surviving mice were re-challenged with prv on day (fig. ) . the mice survival rate was recorded for days. protected against prv infection (fig. ) . however, one mouse from the . μg/ml peptide-treated group and nine mice from the . μg/ml group died within days. to further characterize the prevention effect of piscidin against brain damage induced by prv, the brain sections of mice from the control group, prv-infected, and co-injection group were collected respectively and subjected to pathological examination. the blood stasis was observed in the brain of mice from prv-infected group (fig. a) . the brain of mice from co-injection group showed no specific symptom. after days, the surviving mice were re-challenged with prv at × tcid . the groups co-injected with piscidin and prv failed to provide protection with the survival rates dropping to % at the end of the experiment and the mice in the μg/ml peptide treated group all died. we previously reported the proof-of-concept usage of amps as the bactericidal agents against pathogenic bacteria isolated from pig herds [ ] . the five antimicrobial peptides used in this study have been reported to inhibit the growth of various kinds of viruses including hiv, hsv- , hsv- , etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however there has been limited information about the inhibitory activity of these peptides against the swine-origin viruses. considering this, this study evaluated the effect of these peptides against several porcine viral pathogens. three peptides (caerin, piscidin, maculatin) exhibited inhibitory activity against prv, pedv, tgev, prrsv, and rotavirus. plaque reduction assay showed that the prv infection could be inhibited in a dose-dependent manner by direct treatment of the peptides. a previous study indicated that caerin and maculatin could inhibit hiv infection without affecting t-cell viability and that the activity of caerin was more potent than that of maculatin [ ] . in our research, we further tested the activity of caerin and maculatin against prv, pedv and prrsv. compared with maculatin, caerin showed better activity against most of the tested viruses except tgev (fig. ) . the fact that caerin has a longer α-helix than maculatin, which enables caerin to disintegrate the membrane more potently, might explain the better activity of caerin. usually, the bacterial membrane and viral envelope were reported to be the main targets of the amps. for example, hiv envelope integrity was directly disrupted by caerin and maculatin [ ] . however in this study, tgev and prv which both acquired their envelopes from pk- , exhibited different levels of sensitivity to the tested peptides. in addtion to its envelope, physical characteristics of the virus such as viral size, surface area, and morphology might affect its sensitivity. hnp- also demonstrated a -fold difference in activity against different enveloped viruses [ ] . it should also be noted that the antiviral activity of the amps tested in this study is only observed at concentrations that are already somewhat cytotoxic at h and could be even more so at h or longer. to our surprise, piscidin, caerin, and maculatin a b fig. piscidin protects mice from prv-induced death. a. processed brain sections from control, co-treated, and prv infected groups were subjected to haematoxylin and eosin (h&e). the representative h&e results were shown. b. the mice injected with piscidin or prv, co-treated with piscidin and prv were divided into different groups. surviving mice were challenged with prv on day again. survival status of control and experiment groups were monitored on a daily basis for days (n = ). in the group of piscidin ( μg/ml), mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mice were heavily infected and died on day . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mouse was heavily infected and died on day . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( μg/ml) + prv, mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; in the group of piscidin ( . μg/ml) + prv, mice were heavily infected and died on day . and another mice with severe neurological symptoms were euthanized respectively for animal welfare reasons; in the group of piscidin ( . μg/ml) + prv, mice were heavily infected and died on day and . and another mice with severe neurological symptoms were euthanized for animal welfare reasons; a total of mice from all groups survived and were euthanized by the end of the experiment could consistently inhibit the growth of rv, a non-enveloped rna virus. among these three peptides, piscidin showed the strongest inhibitory activity. it has been reported that piscidin possessed significant antiviral activity to fight against both frog virus and channel catfish virus [ ] . the reduction of fv infectivity resulting from the application of piscidin is due to its capacity to interact with the essential lipid membrane of fv [ ] . based on these results, it could be speculated that there might be other targets for these peptides in addition to the viral envelope, which need further exploration. lactoferricin b and indolicidin showed weak or no activity against the five tested viruses compared with caerin, maculatin, and piscidin. robinson et al reported that the ic of indolicidin against hiv- ranged from μg/ml to μg/ml [ ] . the weak activity of indolicidin against the tested viruses in this study was probably due to the low concentration we used ( μg/ml). it has been reported that amps could inhibit both extracellular and intracellular viruses [ ] [ ] [ ] . our plaque reduction assay result indicated that piscidin, caerin, and maculatin could inhibit prv by directly interacting with the virus. addition of the peptides pre-or post-infection could not help inhibit the prv infection. vancompernolle et al reported that caerin inhibited hiv infection by disrupting the virion envelope [ ] . chinchar et al also reported that amphibian-origin esculentin- p (e p) and ranatuerin- p (r p) could inactivate the channel catfish virus (ccv) by directly interacting with the virions [ ] . the re-emergence of pseudorabies in china since has caused a huge economic loss to the pig farms. three prv strains, i.e. ea, and hnxx, were chosen for further evaluation of peptide's antiviral activity. the peptides inhibited replication of the three prv strains. this is of great importance in fighting against the mutant viruses resulting from natural selection or antibodyinduced events. on the other hand, these peptides had some cytotoxicity on pk- cells (fig. ) . piscidin has been reported to have antiviral and antibacterial properties in vitro [ , ] . however, this had not been tested in vivo. in this study, the data showed that coinjection of prv with piscidin at the concentration of above μg/ml could offer protection against prv infection. even when the concentration of piscidin decreased to . μg/ml, the survival rate of mice could reach %. huang et al previously reported that th - , as an amp isolated from tilapia, could offer % protection against jev infection at μg/ml [ ] . compared with th - , piscidin was more effective even at low concentration. as huang et al noted, th - treated mice surviving from the first jev infection remained alive after second challenge with jev at day [ ] . however, our in vivo studies exhibited a different result. the possible reason needs further exploration. this study indicated that piscidin, maculatin and caerin could inhibit the infection of prv, prrsv, pedv, tgev and rotavirus. among the peptides examined in this study, piscidin showed the strongest antiviral activity against prv both in vitro and in vivo. and it could block the prv-induced cell apoptosis as well. abbreviations amp: antimicrobial peptide; prv: pseudorabies virus; tcid : tissue culture infective dose assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction s domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets new variants of porcine epidemic diarrhea virus, china antimicrobial peptides: do they have a future as therapeutics? in: antimicrobial peptides defensins: antimicrobial peptides of innate immunity antimicrobial peptides: promising alternatives in the post feeding antibiotic era the immunology of host defence peptides: beyond antimicrobial activity inhibition of hiv infection by caerin antimicrobial peptides the chemistry and biological activities of peptides from amphibian skin secretions generation of a dual-target, safe, inexpensive microbicide that protects against hiv- and hsv- disease a review of the design and modification of lactoferricins and their derivatives defensins at the mucosal surface: latest insights into defensin-virus interactions generation and characterization of ul null pseudorabies virus variant in vitro and in vivo role of the pseudorabies virus gi cytoplasmic domain in neuroinvasion, virulence, and posttranslational n-linked glycosylation vaccines against pseudorabies virus (prv) caerin . suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus the use of the antimicrobial peptide piscidin (pcd)- as a novel anti-nociceptive agent single amino acid substitutions at specific positions of the heptad repeat sequence of piscidin- yielded novel analogs that show low cytotoxicity as well as in vitro and in vivo anti-endotoxin activity broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin b a crispr/cas and cre/lox system-based express vaccine development strategy against re-emerging pseudorabies virus establishment and clinical application of a multiplex reverse transcription pcr for detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine group a rotavirus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway porcine epidemic diarrhea virus orf gene prolongs s-phase, facilitates formation of vesicles and promotes the proliferation of attenuated pedv the ubiquitin-proteasome system is required for the early stages of porcine circovirus type replication antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides anti-hiv- activity of indolicidin, an antimicrobial peptide from neutrophils antiviral peptides targeting the west nile virus envelope protein novel influenza virus ns antagonists block replication and restore innate immune function fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors inactivation of frog virus and channel catfish virus by esculentin- p and ranatuerin- p, two antimicrobial peptides isolated from frog skin piscidin: antimicrobial peptide of rock bream modulation of the immune-related gene responses to protect mice against japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin - publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. availability of data and materials data and materials are available upon request by the corresponding author.ethics approval and consent to participate all of the animal experimental protocols were reviewed and approved by the ethics committee of huazhong agricultural university. not applicable. the authors declare that they have no competing interests. key: cord- - iwljdoi authors: chen, qin; li, jian; fang, xue-en; xiong, wei title: detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: iwljdoi a conserved nucleic acid fragment of the nucleocapsid gene of swine transmissible gastroenteritis coronavirus (tgev) was chosen as the target, six special primers were designed successfully. loop-mediated isothermal amplification (lamp) was developed to detect the tgev by incubation at °c for h and the product specificity was confirmed by hphi digestion. standard curves with high accuracy for tgev quantization was constructed by adding × sybr greeni in the lamp reaction. the assay established in this study was found to detect only the tgev and no cross-reaction with other viruses, demonstrating its high specificity. by using serial sample dilutions as templates, the detection limit of lamp was about pg rna, times more sensitive than that of pcr and could be comparable to the nest-pcr. swine transmissible gastroenteritis coronavirus (tgev), as a member of the coronaviridae, is a kind of single-stranded rna virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry. the traditional detection methods, including virus isolation, virus immunodiagnostic assays and pcr tests have the shortcomings, such as precise instruments requirement, elaborate result analysis demand, high cost, long detection time and so forth, which prevent these methods from being widely used [ ] [ ] [ ] [ ] . loop-mediated isothermal amplification (lamp) is a novel nucleic acid amplification method, which amplifies dna/rna with high specificity, sensitivity and rapidity under isothermal condition [ ] . it has already found wide application in rna virus detection, such as foot-and-mouth disease virus [ ] , swine vesicular disease virus [ ] , taura syndrome virus [ ] , severe acute respiratory syndrome coronavirus and h n avian influenza virus [ , ] . in this study, lamp method was applied in developing qualitative and quantitative detection system of tgev, while its specificity and sensitivity were assessed. swine transmissible gastroenteritis coronavirus (tgev, strain h), porcine reproductive and respiratory syndrome virus (prrsv), pesudorabies (prv), porcine parvovirus (ppv) derived from their passages in cell culture were provided by shanghai entry-exit inspection and quarantine bureau (shciq); nucleic acids of footand-mouth disease virus (fmdv) and classical swine fever virus (csfv) were obtained from chinese academy of inspection and quarantine (caiq). total genomic rna was extracted using trizol kit (invitrogen, usa). dna was extracted by dna blood mini kit (qiagen, germany). after elution in μl nuclease-free water, rna/dna samples were stored at - °c before use. the original concentration of rna/ dna sample was about ng/μl. complete genome sequences of fifteen different tgev strains/isolates and nine other similar viruses were obtained from genbank, and the homology was analyzed using the vector nti. the conserved fragment with high homology was chosen as the target region which and used to design the tgev lamp primers by the primer explorer v software http://primerexplorer. jp/e/. the target rna of tgev was first reverse transcripted using superscript™ ii (invitrogen, usa) and then amplified by pfu dna polymerase using forward primer: ggaagagaactgcaggtaa and reverse primer: ccatcttcctttgaagtcca. the amplified product was purified from agarose gels and then cloned into e. coli jm using the pmd -t vector. the target plasmid with the original concentration of . × copies/μl was extracted by the plasmid mini preparation kit and identified by the nm absorption spectroscopy, which was then used as the standard for the quantitative analysis. the amplified products were digested with hphi to confirm its specificity. the result of tgev-lamp was analyzed by agarose gel electrophoresis and fluorescence by adding × sybr greeni in the lamp reaction. the specificity of tgev-lamp was examined by the use of rna (or dna) extracted from five other pig disease viruses. the sensitivity of tgev-lamp was evaluated by comparing with pcr [ ] and nest-pcr[ ], using -serial tgev rna dilutions ( - to - ) as templates. lamp primers were designed using the primer explorer v software based on a conserved fragment of the nucleocapsid gene (fig. ) . the primers including outer primers (f and b ), inner primers (fip and bip) and loop primer (lf and lb) were shown in table . tgev deprived from the cell culture was first qualitatively analyzed by lamp. amplification products were analyzed by agarose gel electrophoresis. as shown in fig. (lane ), amplification could be carried out at °c and showed a ladder-like pattern on the gel while the negative control gave no bands (lane ). the specificity of the lmap product was confirmed by hphi digestion. predictable product of the -bp motif was resolved on the gel as theoretical expected (fig. , lane ) . standard control was used to develop real-time fluorescence lamp for quantitatively analyzing tgev. dynamic curves for tgev quantification was generated by serially diluting the standard control from . × to . × copies/μl (fig. ) . the log linear regression plot (standard curves) was obtained by plotting the time-to-positive (ttp) values against genome copies. the correlation coefficients were . (fig. ) five other pig viruses were used to confirm the specificity of the lamp for tgev detection. the results showed that only tgev detected gave amplification products while no amplification available to other viruses (fig. ) . the sensitivity of lamp was demonstrated by comparing with pcr tests using serial dilutions ( - to - ) of tgev rna samples as template. as shown in fig. , lamp and nest-pcr were able to detect - dilution (about pg rna), whereas pcr could only amplify the - dilution. therefore, the sensitivity of tgev-lamp could be comparable to nest-pcr, -fold higher than pcr. it is very important to find out a conserved nucleic acid fragment for designing specific lamp primers and developing efficient, accurate lamp assay [ , ] . in this study, the nucleic acid sequence homology of tgev strains/isolates and other similar viruses available from genbank were analyzed by vector nti software. the most conserved fragment of bp was found in the nucleocapsid protein gene which showed highly homology among different tgev strains/isolates (more than %) and low homology among other similar viruses (less than . %). the tgev lamp primers targeting the conserved sequence were designed successfully by the primer designer v software. the target region was amplified successfully by the lamp with a characteristic ladder-like pattern of bands from bp on the gel. this is because the final products of lamp are a mixture of stem-loop dnas with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand [ , ] . after digestion with hphi, -bp motif was resolved on the gel as expected, demonstrating the specific structure of amplification products, which could also be validated by nucleic acid sequencing. as a kind of nucleic acid amplification method, lamp could not only qualitatively detect the tgev, but also quantitatively analyze the virus. in this study, real time fluorescence lamp for quantitatively detection of tgev was established by adding × sybr greeniin the lamp reaction. three standard curves established by tgev standards displayed the good correlation between the ttp and virus copies, implicating the great potential in quanlitatively detecting tgev. five other viruses were used in this study to confirm the specificity of lamp. the results showed no amplification in all viruses tested, which makes the lamp more accurate and reliable for tgev detection. the high specificity of lamp is most probably attributable to recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences during the second reaction stage. the sensitivity of lamp was evaluated using various tgev-rna dilutions as templates. the assay exhibited almost equivalent sensitivity to the nest-pcr and -fold higher than the pcr, indicating that the lamp is a more powerful diagnosis tool to detect tgev in lower copy conditions [ ] . in addition, the tgev-lamp developed has advantages in its rapid detection and simple operation. the only equipment required for the reaction is a water bath or heat block. the assay developed is a faster detection method for the tgev detection, only taking about h, which means the whole diagnosis including rna extraction, amplification and product detection could be completed within one and a half hour after receiving of the samples. it is anticipated that with the advantages of specificity, sensitivity, reliability, rapidity and easy manipulation, lamp will turn out be a powerful molecular tool for the tgev detection in practice. in conclusion, this study demonstrates that the lamp method established could detect only the tgev and no cross-reaction with other viruses, the detection limit was about pg rna, which was times more sensitive than that of pcr and could be comparable to the nest-pcr. virus isolation and serum antibody-responses after infection of cats with transmissible gastroenteritis virus -brief report use of monoclonal antibodies in blocking elisa detection of transmissible gastroenteritis virus in faeces of piglets an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus loop-mediated isothermal amplification of dna novel reverse transcription loopmediated isothermal amplification for rapid detection of foot-andmouth disease virus a: one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus rapid and sensitive detection of taura syndrome virus by reverse transcription loop-mediated isothermal amplification development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific figure standard curves of real-time fluorescence tgev-lamp negative control. figure sensitivity of lamp for tgev detection. a, lamp; b, pcr; c, nest-pcr the authors declare that they have no competing interests. key: cord- -yrqpq cf authors: jevšnik, monika; steyer, andrej; zrim, tamara; pokorn, marko; mrvič, tatjana; grosek, Štefan; strle, franc; lusa, lara; petrovec, miroslav title: detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: yrqpq cf background: human coronaviruses (hcovs) are a well-known cause of respiratory infections but their role in gastrointestinal infections is unclear. the objective of our study was to assess the significance of hcovs in the etiology of acute gastroenteritis (age) in children < years of age. methods: stool samples and nasopharyngeal (np) swabs collected from children hospitalized for age ( also had respiratory symptoms) and otherwise healthy control children admitted for elective surgery were tested for the presence of four hcovs using real time rt-pcr. registered at clinicaltrials.gov (reg. nct ). results: hcovs were more frequent in patients with age than in controls ( / , . % versus / , . %; odds ratio, or . ; % confidence interval, ci . – . ; p = . ). three of four hcov-positive members in the control group, asymptomatic when sampled, recalled gastrointestinal or respiratory symptoms within the previous days. in patients with age, hcovs were present in np samples more often than in stools ( / , . %, versus / , . %; p = . ). in / children with hcovs detected in stools, the viruses were also detected in np swabs. patients had a significantly higher probability of hcov detection in stool (or ; % ci . – . ; p = . ) and also in stool and/or np (or . , % ci . – . ; p = . ) than healthy controls. all four hcovs species were detected in stool and np samples. conclusions: although hcovs were more frequently detected in patients with age than in the control group, high prevalence of hcovs in np swabs compounded by their low occurrence in stool samples and detection of other viruses in stool samples, indicate that hcovs probably play only a minor role in causing gastrointestinal illness in children < years old. human coronaviruses (hcovs), known since the late s, have been recognized as a frequent cause of mild respiratory tract infections (rtis) and occasionally as a potential cause of severe lower rti in premature infants and children with underlying diseases [ ] . their role in enteric infections is less clear. even though coronavirus-like particles have been seen by electron microscopy in stool samples from patients with diarrhea, they have been also found in healthy individuals [ ] . however, coronaviruses are associated with diarrheal disease in several animal species. thus, a dual enteric and respiratory tropism has been reported for the bovine coronavirus causing winter dysentery in calves [ ] . interest in coronaviruses in relation to enteric diseases in humans increased with the emergence of severe acute respiratory syndrome (sars) and identification of sars-cov in [ , ] . sars was characterized clinically by signs/ symptoms of severe lower rti but several patients also had gastrointestinal complaints [ , ] . diarrhea has also been a prominent clinical feature in several patients with respiratory infection caused by other hcovs [ ] [ ] [ ] . furthermore, all hcov species were recently detected in stool samples of patients with acute gastroenteritis (age) by modern molecular methods, but their role in human gastrointestinal infections remains uncertain [ ] [ ] [ ] . in previous studies, hcovs were demonstrated in patients with gastrointestinal and/or respiratory involvement, but in none of these studies was concurrent collection of stool and respiratory samples performed. the main objective of the present study was to evaluate the presence of hcovs in simultaneously collected stool samples and nasopharyngeal (np) swabs in children with age (with or without associated respiratory symptoms) and in control subjects, with the aim of appraising their role in the etiology of age. from october to september , children < years of age were referred to the department of infectious disease with the diagnosis of age. of them were enrolled in the study. the others did not meet inclusion criteria for age ( children), were not enrolled because their parents did not consent with the inclusion in the study ( children) or because they did not pass stool during hospital stay ( children). the median age of included children was . months. the female: male subject ratio was : . ( / ; . % girls). in of the subjects ( . %), signs and symptoms were limited to the gastrointestinal tract, while ( . %) also had signs and symptoms compatible with acute rti such as rhinitis, pharyngitis, conjunctivitis, otitis or bronchiolitis. in the group of children with age, both samples (stool sample and np swab) were obtained from / patients (only stool sample was acquired from four of them). from children comprising the healthy control group, both samples were available from patients, only a stool sample from one child and only a np swab from six. samples obtained at follow-up examination days after initial testing were available for / ( . %) children with age. unfortunately, remaining children did not respond and did not come to follow up visit days after acute illness. children who did not come to the follow up visit had similar clinical characteristics as those who attended the follow up visit. therefore, authors assumed that they were representative of the entire study population. in of the patients, both stool and np swab samples were obtained, while only stool sample was obtained from nine children and solely np swab was available from nine others. hcovs were detected in / stool samples from children with age ( . %; % ci . - . %)-among which / were detected also in np samples-and in / np swabs ( . %, % ci . - . %). hcovs were more often detected in np swabs than in stool samples (p = . by mcnemar's test). four of six ( %, % ci - %) children with hcov-positive stool samples had symptoms of respiratory and gastrointestinal infection ( table ) . of the six hcov-positive stool samples, three were characterized as oc , one as nl , one as hku and one as e. in two ( . %) stool samples hcov (one e and one oc ) was detected as a single pathogen, while in four samples the presence of other viruses was also established (table ) . of positive np samples, ( %, % ci - %) originated from children with age associated with rti. the species distribution was as follows: nine oc ( %), seven hku ( %), three e ( %) and three nl ( %; table ). fifteen ( . %) of hcovs positive np swabs were detected as a single viral pathogen; also in this subgroup the most common was oc ( / , . %). hcovs was detected as a single viral pathogen in np swab in / ( . %) children with age as the only clinical sign and in / ( . %) np swabs of children who had age associated with signs/symptoms of rti (or = . ; % ci . - ; p = . ). hcovs were not detected in any of follow-up stool samples. of follow-up np swabs, ( . %, % ci . - . %) were positive for hcovs: eight were typed as hku , four as nl and two as oc ; one positive sample remained untyped because of the low viral load. in of positive children, hcovs were demonstrated only in the follow-up sample, while in five children the presence of hcovs was established in the first and follow-up np swab; four of these also had hcovs in stools at the first sampling (table ). at the time of follow-up testing, / children ( %, % ci . - . %) with hcov-positive np swab had gastrointestinal signs/symptoms; / ( %, % ci - %) had rti and four ( . %, % ci - %) were asymptomatic. none of the samples from children positive for hcovs in np swabs and/or stool sample had viruses detected in blood sample taken simultaneously. hcovs were detected in / stool samples ( . %, % ci - . %) and in / np swabs ( . %, % ci . - . %) from the control group. the hcov from the stool sample remained untyped whereas respiratory samples revealed one nl , one e and one hcov that was untyped. hcovs remained untyped because of the low viral load. all children included in the control group were asymptomatic on the day of sampling; however a detailed history showed that three out of four children who were found to be hcov positive reported symptoms indicating respiratory or gastrointestinal infections within days before sampling. patients with age had a significantly higher probability of hcov detection in stool samples than controls (or ; % ci . - . ; p = . ), while a weaker association was observed using np samples (or . ; % ci . - . ; p = . ). if positive stool samples and/ or np swabs were considered together, the association remained statistically significant (or . ; % ci . - . ; p = . ). hcovs are recognized as causes of respiratory infections but their role in gastrointestinal infections has not been clarified. in the present study we assessed the significance of hcovs in the etiology of age in children < years old by testing samples from stools and np swabs for the presence of four hcovs by rt-pcr. in the patients with age, follow-up sampling was performed days after the initial testing and we acquired detailed clinical information for each participant. our study revealed that hcovs were present in patients with age and that viruses were more prevalent in the patients than in the control group ( / , . % versus / , . %, or . , % ci . - . %, p = . ). moreover, the higher probability of hcov detection in the stool samples of patients with age than among controls (or ; % ci . - . ; p = . ) indicated a causal association of hcovs with age. this was supported by the observation that three of four hcov-positive members of the control group, who were asymptomatic at the time of sampling, reported symptoms within a day period before sampling. however, in patients with age one would think that the etiologic agent is more likely to be demonstrated predominantly in stool samples, whereas in our study hcovs were detected more often in np swabs than in stools ( / , . % versus / , . %; p = . ). in addition, in five of six children with hcovs detected in the stool samples, the viruses were also demonstrated in np swabs. there are several partial explanations for this. the course of the events in hcovs infection might have been similar to those known to be operative in animals and as suggested for sars-cov [ , ] . thus, fecal-oral transmission is followed by intestinal damage, leading to viremia and respiratory infection. however, we tested all patients with hcovs found in stool samples or np swabs for the presence of the viruses in blood but did not get any positive result. in addition, viremia usually results in the involvement of the lower rti whereas upper rti is usually the result of direct spread of infection through neighboring tissues. although we cannot exclude viremia that was missed in our patients, it seems more likely that hcovs did not cause viremia. the other possibility is that oral transmission of hcovs is followed by gastrointestinal infection in patients with age and in the majority of children by symptomatic or asymptomatic np infections because of the direct spread of viruses from the oral cavity to the nasopharynx, or secondarily from the gastrointestinal region from vomiting ( . % of our children experienced vomiting in addition to diarrhea). because enveloped hcovs viruses are not stable in stools, their persistence is much more limited and transient than in the upper respiratory tract. this could explain the lower detection rate of hcovs in stools than in np swabs. the third possibility is that the primary location of hcovs replication is in the nasopharynx, resulting in asymptomatic or symptomatic respiratory infections, and that the hcovs occasionally found in stool samples had their origin in the respiratory system but were passed down the alimentary tract. this theory would enable an elegant explanation for the higher proportion of np swabs positive for hcovs than in stools and for the finding that all but one patient with hcovs in the stool samples also had them present in np swabs, but does not explain age, which should have been caused by other agent. the theory is further supported by the finding that only in two children hcovs were found as a sole viral agent in stool while in four cases the presence of other viruses known to cause age was also established. surprisingly, hcovs were detected as a single viral pathogen in / ( . %) np swabs from children with age without concomitant respiratory symptoms. more than half ( / , . %) of children with age also had respiratory symptoms at the time of sampling. this proportion was similar for patients with hcovpositive np swabs ( . %) and for those with hcovpositive stool samples ( . %), whereas it was higher ( %) for children with hcovs detected in both samples. however, there were very few patients with hcovs in stool samples and with viruses found in both sources (six and five, respectively) and the difference was not statistically significant. in a report from finland, % of children with age and hcov-positive stool samples had respiratory symptoms at the time of sampling, while in a study from the usa both symptoms appeared in % of children [ , ] . the median duration of illness and of hospitalizations were similar for hcovs positive and negative children (table ) , and the differences were not statistically significant (duration of illness: p = . for stool samples, p = . for np swabs, duration of hospitalization: p = . for stool samples and p = . for np swabs). no significant difference was found between the proportion of hcovs present at the time of acute illness information on the occurrence of hcovs in healthy persons is limited. our finding that hcovs were rare in stool samples from healthy children ( . %, % ci - . %) and in np swabs ( . %, % ci . - . %) is consistent with a report finding hcovs in only . % of stools from healthy children [ ] . all four hcovs species were detected in stool and np samples; the most common was oc ( % in stool samples, % in np samples). e occurred only in children with age without respiratory symptoms, whereas nl was found only in children with rti as an additional sign. in conclusion, the presence of hcovs in patients with age, the more frequent demonstration of these viruses in patients than in the control group and the higher probability of hcovs detection in the stools of patients with age than in controls indicates some causal association of hcovs with age. however, interpretation of the role of hcovs in age is complex because the viruses were more often demonstrated in np swabs than in stool samples, because of detection of other viruses known to cause age in stool samples and because more than half of the children with age had also signs and symptoms of rti. the findings of the present study suggest that hcovs probably played only a minor role in gastrointestinal illness in these children. this was a part of a prospective study on viral respiratory and gastrointestinal infections in children < years. children in this age group who had been admitted to the department of infectious diseases of the university medical centre ljubljana, from october to september , with the diagnosis of age (defined as the passage of three or more loose or liquid stools in h) were eligible for the study. stool specimens, np swabs and blood samples were obtained at admission and at a followup visit days after the initial sampling. children in the same age group admitted to the department of pediatric surgery and intensive care for planned elective surgical procedures (i.e. inguinal hernia repair, testicular retention or hydrocele) were selected as a healthy control group. only children without infections in last four weeks preceding the surgery were eligible for surgery admission. all children were clinically examined and only those without any symptoms of infection were admitted. approximately one third of the admitted children have not been included due to parents' refusal of entering the study or due to logistical problems with sample collection. in this group, stool samples and np swabs were collected at admission. at this time, information was obtained on the presence of signs and symptoms compatible with gastrointestinal and/ or respiratory infection within the last days. to minimize children's discomfort nasopharyngeal sampling was performed after patients underwent anesthesia. the study protocol was approved by the national medical ethics committee of the republic of slovenia (no. / / ). written informed consent was obtained from all parents of participating patients and control subjects. the principles of the helsinki declaration, the oviedo convention on human rights and biomedicine and the slovene code of medicine deontology were strictly followed in the conduct of this research. the study protocol was also submitted to clinicaltrials.gov registry with the title viral respiratory and gastrointestinal infections in children under years of age (reg. nct ). stool samples were diluted in sterile phosphate-buffered saline (pbs) to a % suspension. aliquots of μl of magna pure bacteria lysis buffer (roche applied science, mannheim, germany) and μl of proteinase k (qiagen, hilden, germany) were added to μl of stool suspension. np swabs were collected using flocked-tip swabs and transported to the laboratory in the copan universal transport medium (utm-rt) system (copan italia, brescia, italy). before the extraction procedure, μl of equine herpesvirus and μl of equine arthritis virus isolates were added to all samples. specific target sequences of these viruses were subsequently amplified in separate real time reverse transcription polymerase chain reactions (rt-pcrs) as an internal control to ensure that negative results were not caused by poor nucleic acid extraction or inhibition of the rt-pcr assay [ , ] . the initial volume used for extracting total nucleic acids was μl of stool suspension, μl of vigorously vortexed np swab medium and μl of whole blood samples. nucleic acids were extracted using total nucleic acid isolation kits on a magna pure compact instrument (roche applied science), according to the manufacturer's instructions. coronaviruses including hku , nl , e and oc were detected in stool samples, np swabs and whole blood samples by molecular methods using primers and probes as described by kuypers et al. [ ] . for amplification of - bp fragments of the polymerase b gene of coronaviruses, a one-step real-time rt-pcr assay was used in a stepone real-time pcr system (applied biosystems, carlsbad, ca). briefly, μl of total nucleic acid was added to μl of reaction mixture including × reaction mix, superscript w iii rt/platinum w taq mix (invitrogen, carlsbad, ca) with an additional mm mgso . the cycling conditions were as follows: min at °c, min at °c and cycles of s at °c and s at °c. all np swabs and stool samples in which hcovs were established were also tested for the presence of other viruses. in np swabs respiratory syncytial virus (rsv), influenza viruses a and b (flu a-b), parainfluenza viruses - (piv - ), metapneumovirus (hmpv), human bocavirus (hbovs), adenovirus (adv) and rhinovirus (hrv) [ ] [ ] [ ] [ ] [ ] [ ] [ ] were searched for by real-time rt-pcr. stool samples were tested for the presence of hbovs and adv using molecular methods as described previously [ , ] , and for other gastroenteric viruses by electron microscopy (em). the presence of norovirus genogroups i and ii and astrovirus was ascertained by real-time rt-pcr [ , ] , while group a rotavirus and adenovirus type / were detected by antigen-elisa premier rotaclone and premier adenoclone (meridian bioscience, cincinnati, oh). data are reported as the frequency (percentage) and % confidence intervals (cis) for proportions obtained using exact binomial tail areas. mcnemar's test was used to compare the frequency of hcov positivity of patients at baseline and after days of follow-up. univariate logistic regression models with firth's correction were used to assess the association between hcov positivity and study group (patient or control) and the results are reported as the odds ratio (or) with % ci and two-sided p values. mann-whitney test was used to compare the median duration of illness and of hospitalization of hcovs positive and hcovs negative children. the tecumseh study of respiratory 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transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages real-time pcr assays for detection of bocavirus in human specimens simultaneous detection of human bocavirus and adenovirus by multiplex real-time pcr in a belgian paediatric population diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr novel approach for detection of enteric viruses to enable syndrome surveillance of acute viral gastroenteritis submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution hcovs: human coronaviruses; np swab: nasopharyngeal swab; rti: respiratory tract infections; age: acute gastroenteritis. the authors declare that they have no competing interests. this study was supported by the slovenian research agency (research program p - ) and institutional department funds. key: cord- -lakdi x authors: kang, xiao-ping; jiang, tao; li, yong-qiang; lin, fang; liu, hong; chang, guo-hui; zhu, qing-yu; qin, e-de; qin, cheng-feng; yang, yin-hui title: a duplex real-time rt-pcr assay for detecting h n avian influenza virus and pandemic h n influenza virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: lakdi x a duplex real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay was improved for simultaneous detection of highly pathogenic h n avian influenza virus and pandemic h n ( ) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. the sensitivity of this duplex real-time rt-pcr assay was . tcid( )( % tissue culture infective dose) for h n and . tcid( )for the pandemic h n , which was the same as that of each single-target rt-pcr for pandemic h n and even more sensitive for h n with the same primers and probes. no cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. two hundred and thirty-six clinical specimens were tested by comparing with single real-time rt-pcr and result from the duplex assay was % consistent with the results of single real-time rt-pcr and sequence analysis. in march and april , a novel swine-origin pandemic h n influenza virus appeared and spread worldwide. the pandemic of h n influenza virus poses a public health threat. according to the world health organization (who), as of april , worldwide at least death cases have been reported [ ] . compared with influenza a h n virus, the highly pathogenic avian influenza virus h n has a much higher mortality rate: h n has a mortality rate of more than % while novel pandemic h n has only about % [ ] . nevertheless, pandemic h n and highly pathogenic h n manifest similar clinical symptoms at the early stage of infection [ ] . early detection of these two pathogens is an essential prerequisite for effective control and prevention of the pandemic. real-time rt-pcr/pcr is a powerful method for the sensitive and specific detection of virus-derived nucleic acids in clinical samples [ ] . it is time-saving and more specific compared with endpoint pcr. therefore, realtime rt-pcr assay has been widely used and recom-mended by who for many rapid viral pathogen detection. moreover, multiplex assays allow to measure several fluorophores in one well, which can simultaneously detect different target sequences [ ] . since the outbreak of pandemic h n influenza, many real-time rt-pcr assays have been developed for detecting of the novel h n [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some multiplex real-time rt-pcr assays for simultaneous typing (a/b) and subtyping of h , h , h , h , h and h of influenza a viruses have also been reported [ ] [ ] [ ] [ ] . however, no studies were reported for subtyping of the novel pandemic h n and h n simultaneously. in this study, a duplex taqman real-time rt-pcr assay was improved by adjusting the concentrations of primers and probes in the who protocols. the assay could simultaneously detect h n avian influenza virus and pandemic h n influenza virus, which could be used for early diagnosis of influenza-like patients and for epidemiological surveillance. the primers and probes for the novel pandemic h n and the influenza h n were derived from the protocols recommended by who [ , ] . the sequence of primers for h n were h -sense: '-gga act tac caa ata ctg tca att tat tca- ', h -antisense: '-cca taa aga tag acc agc tac cat ga- ' and h -probe: '-* correspondence: yangyinhui@hotmail.com state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology, beijing , china † contributed equally full list of author information is available at the end of the article hex-ttg cca gtg cta ggg aac tcg cca c-bhq - ' which was labeled with the reporter hexachloro- -carboxyfluorescein (hex) at the ' end and a black hole quencher (bhq ) at the ' end [ ] . the sequence of primers and probe for pandemic h n were h -sense: '-gtg cta taa aca cca gcc tyc ca- ', h -antisense: '-cgg gat att cct taa tcc tgt rgc- ', h -probe: '-fam-cag aat ata cat* ccr gtc aca att gga raa- ' which was labeled with the reporter -carboxyfluorescein ( -fam) at the ' end and a black hole quencher (bhq ) at the a modified t residue [ ] . all primers and probes were synthesized by dalian takara company. rna was extracted from the supernatants of cultured viruses or from clinical specimens by rneasy mini kit (qiagen, hilden, germany) according to the manufacture's instruction. virus rna extractions were conducted in biosafety level (bsl- ) facilities. the duplex real-time rt-pcr amplification was carried out in a μl volume reaction with the quantitect probe rt-pcr kit (qiagen, hilden, germany) with the lightcycler . system (roche, mannheim, germany). different concentrations of the primers and probes for both h n and h n were combined and adjusted to improve the sensitivity of the assay [ ] . the concentrations of primers and probe for h gene in the duplex system was optimized by using a serial of -fold diluted h n rnas as templates. the sensitivity of the duplex assays was evaluated at the concentration of h primers as . μm, . μm, . μm and . μm. the results showed that the sensitivity of the duplex assays dramatically increased with the concentration of h primers. the duplex assay had the best detecting result at the h primer concentration of . μm. the concentration of probes for h gene was also optimized by examining the concentration at . μm, . μm, . μm and . μm, and any significant different results at different concentrations was not observed. therefore, . μm was selected as the final concentration of h probe in the duplex realtime rt-pcr system. similar to the duplex assay for h gene, the concentrations of primers and probe for h also were optimized by using rna from , . , . and . tcid h n as templates. the concentration of h primers was optimized from . μm, . μm, . μm, . μm and . μm, while the concentration of h probe was optimized at . μm, . μm, . μm and . μm. thus, . μm of primers and . μm of probe were selected as the optimal concentration for h gene. the reactions were incubated at °c for min, followed by °c for min, cycles of °c for s, and °c for min. fluorescence was recorded at °c. the final optimized reaction mixture consisted of μl of × reaction buffer, . μl reverse transcription enzyme, . μm of each h n primer, nm of each novel h n primer, nm of h n probe, nm of pandemic h n probe and μl rna templates. the protocol of real-time rt-pcr for influenza a (h n ) recommended by who used a concentration of nm each of novel sw h primers and nm of sw h probe [ ] . the who real-time rt-pcr protocol for h n used a concentration of nm each of h primers and nm of h probe [ ] . the sensitivity of the single and duplex real-time rt-pcr for novel h n and h n virus was evaluated by -fold diluted virus rna, respectively. the pandemic h n influenza virus strain a/beijing/ / (h n ) (genbank: gq -gq ) and avian influenza virus h n strain a/beijing/ / (genbank: ef -ef ) were cultured on mdck cells and tested by the assay. the analysis of threshold cycles (ct) signals as a function of log tcid titers of tested viruses showed a nearly linear decrease of ct value with increased virus titer. the sensitivity of the single realtime rt-pcr assay was . tcid for both novel pandemic h n and h n virus. the detection threshold of the duplex real-time rt-pcr assay was . tcid for the pandemic h n and . tcid for h n virus. compared with single real-time rt-pcr assay, the duplex real-time rt-pcr assay had the same sensitivity for pandemic h n virus, and about -fold more sensitive for h n virus ( . tcid ) (figure ). this was likely because the procedure of the duplex assay for h n was optimized for roche lightcycler . system, while the single real-time rt-pcr assay recommended by who was optimized for abi instruments [ ] . the specificity of the duplex real time rt-pcr assay was validated by using human genomes and a panel of respiratory tract viruses including human seasonal h n influenza viruses, seasonal h n virus, human respiratory syncytial virus a and b, human coronavirus e, human coronavirus oc , influenza b virus, human parainfluenza virus and human adenovirus. all samples were tested negative (data not shown). a total of clinical throat swab specimens from suspected cases of novel pandemic h n patients were collected. the samples were first detected by who realtime rt-pcr protocol for influenza a (h n ). as the who protocol also included a set of primers for universal detection of type a influenza viruses, thus, only the influenza a positive samples confirmed by who protocol were further tested by the duplex system for comparison with the results of who protocol. among the influenza type a positive specimens, specimens were positive for the pandemic h n and none for the h n tested by the duplex system. duplex real-time rt-pcr assays showed % coincident results with the single real-time assays. sequence analysis was also con-ducted with samples selected randomly from the novel h n positive specimens. since few clinical specimens was available currently in china, a mock clinical h n specimen was extracted from the spleen of balb/c mice infected with high pathogenic h n influenza virus to validate the specificity of high pathogenic h n virus. the duplex real-time rt-pcr assay showed strong positive result, which was confirmed by single real-time pcr and subsequent sequencing analyses. this demonstrated that the duplex real-time rt-pcr assay was a substitute method for the current single real-time rt-pcr method. so far, the number of confirmed severe novel h n cases has reached about seven hundred thousand in china and increased quickly. the lessons from and influenza suggested that the pandemic might return in a much more lethal form in the second wave [ ] . recently, the increasing geographic distribution of high pathogenic h n as well as its ability to transfer to humans and to cause severe infection has raised serious concerns regarding the control measures of this virus. the duplex real-time rt-pcr assay for pandemic h n and high pathogenic h n would play an important role for control and prevention of pandemic caused by these viruses. figure sensitivity and dynamic range of the duplex real-time rt-pcr assay for detection of h n and pandemic h n viral rna. serial -fold dilutions of viral rna standard (from × - to × tcid ) were plotted against the threshold cycle (ct). a minimum of . tcid h n viruses or . tcid h n viruses per reaction could be detected. the coefficient of determination (r ) and the equation of the regression curve (y) calculated. who: pandemic (h n ) -update . . . who: cumulative number of confirmed human cases of avian influenza a/(h n ) reported to who comparative analysis between a low pathogenic and a high pathogenic influenza h hemagglutinin in cell entry nitsche a: real-time pcr in virology real-time multiplex pcr assays detection of human novel influenza a (h n ) viruses using multifluorescent real-time rt-pcr switching gears for an influenza pandemic: validation of a duplex reverse transcriptase pcr assay for simultaneous detection and confirmatory identification of pandemic (h n ) influenza virus development of a real-time rt-pcr assay for a novel influenza a (h n ) virus eis-hübinger a, drosten c: detection of influenza a(h n ) virus by real-time rt-pcr rapid sybr green i and modified probe real-time reverse transcription-pcr assays identify influenza h n viruses and distinguish between pandemic and seasonal strains detection of novel (swine origin) h n influenza a virus by quantitative real-time reverse transcription-pcr confirmation of the first hong kong case of human infection by novel swine origin influenza a (h n ) virus diagnosed using ultrarapid, real-time reverse transcriptase pcr one-step real-time rt-pcr for pandemic influenza a virus (h n ) matrix gene detection in swine samples design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by smartcycler real-time reverse transcription-pcr typing (a/b) and subtyping (h /h / h ) of influenza a viruses by multiplex real-time rt-pcr assays a multiplex real-time rt-pcr for detection and identification of influenza virus types a and b and subtypes h and n development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type a including h and h subtypes who: cdc protocol of realtime rtpcr for swine influenza a(h n ) who: recommendations and laboratory procedures for detection of avian influenza a(h n ) virus in specimens from suspected human cases a duplex realtime reverse transcriptase polymerase chain reaction assay for the detection of st. louis encephalitis and eastern equine encephalitis viruses influenza: the mother of all pandemics a duplex real-time rt-pcr assay for detecting h n avian influenza virus and pandemic h n influenza virus virology journal the authors declare that they have no competing interests.authors' contributions xk and tj: designed the study, did laboratory testing, analysed the test results, co-wrote and edited the manuscript. yl, fl, lh and gc took samples and did laboratory testing. qz, cq and yy organized the overall project and helped edit the manuscript. all authors have read and approved the final manuscript. key: cord- -ucm wx b authors: b’krong, nguyen thi thuy chinh; minh, ngo ngoc quang; qui, phan tu; chau, tran thi hong; nghia, ho dang trung; do, lien anh ha; nhung, nguyen ngoc; van vinh chau, nguyen; thwaites, guy; van tan, le; van doorn, h. rogier; thanh, tran tan title: enterovirus serotypes in patients with central nervous system and respiratory infections in viet nam – date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: ucm wx b background: enteroviruses are the most common causative agents of human illness. enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (enterovirus a and d ), and are of interest as emerging viruses. here, we typed enterovirus a-d (ev) from central nervous system (cns) and respiratory infections in viet nam. methods: data and specimens from prospective observational clinical studies conducted between and were used. species and serotypes were determined using type-specific rt-pcr and viral protein or (vp , vp ) sequencing. results: samples from patients with cns infection ( children – csf and respiratory/rectal swabs) and adults ( csf) and respiratory infection ( children – respiratory swabs) were analysed. twenty-six different serotypes of the four enterovirus species (a-d) were identified, including ev-a and ev-d . enterovirus b was associated with viral meningitis in children and adults. hand, foot and mouth disease associated enteroviruses a (ev-a and coxsackievirus [cv] a ) were detected in children with encephalitis. diverse serotypes of all four enterovirus species were found in respiratory samples, including polio-vaccine viruses, but also cv-a and ev-d . with the exception of ev-d , the relevance of these viruses in respiratory infection remains unknown. conclusion: we describe the diverse spectrum of enteroviruses from patients with cns and respiratory infections in viet nam between and . these data confirm the global circulation of enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. enteroviruses are non-enveloped single stranded rna viruses of the genus enterovirus within the family of picornaviridae. seven species of enterovirus are associated with human disease: enterovirus a-d and rhinovirus a-c. while rhinoviruses commonly cause mild respiratory illness, enteroviruses a-d (evs) are a significant cause of morbidity and mortality worldwide. prior to being reclassified as ev a-d, evs were originally classified as polioviruses (pv) - , coxsackieviruses (cv) a - and b - , echoviruses (e) - and numbered enteroviruses ( - ) [ ] . in addition to the surveillance system for poliomyelitis, some countries have established comprehensive surveillance programs for circulating non-polio evs [ , ] . in recent years, surveillance activities have been enhanced in response to the emergence of hand, foot, and mouth disease (hfmd) causing enteroviruses in the asia-pacific region [ ] and the global spread of ev-d causing respiratory infections [ , ] . ev infections are often asymptomatic, but may also result in a diverse spectrum of clinical illness, varying from mild febrile illnesses to severe disease of the cutaneous, gastrointestinal, respiratory, cardiovascular, and central nervous system (cns) [ , ] . generally, ev a is associated with herpangina and hand, foot and mouth disease (hfmd), ev b with herpangina and sporadic and epidemic viral meningitis or encephalitis, ev c with poliomyelitis and ev d with respiratory infections [ , [ ] [ ] [ ] . in viet nam, since , various serotypes of ev a, most commonly enterovirus a (ev-a ), coxsackievirus a (cv-a ), cv-a , and cv-a have been associated with outbreaks of hfmd [ , ] and evs have also been frequently detected in aetiological studies of cns and respiratory infections [ ] [ ] [ ] [ ] [ ] . in the majority of aetiological studies only generic rt-pcr is used for detection of enteroviruses [ - , , ] . information about specific enterovirus serotypes circulation and their associated clinical phenotypes therefore remains sparse. here we report the clinical associations and serotyping results of evs that were previously detected in our studies of cns and respiratory infections in southern and central viet nam between and . data and samples from prospective observational clinical studies on cns (n = ) and respiratory (n = ) infections, conducted in viet nam between and were used [ - , , ] . patients were enrolled according to studyspecific case definitions for cns infection, acute respiratory infection and lower respiratory tract infection. the three cns studies were conducted to determine the etiology of cns infections in children in children's hospital in ho chi minh city [hcmc] ( ), and in adults in the hospital for tropical diseases ( diseases ( - and in a network of provincial hospitals in the southern and central part of viet nam ( ) ( ) ( ) ( ) . in these three studies, csf was obtained from all participants, while throat and rectal swabs were only taken from children. the two respiratory infections studies were conducted to study lower respiratory tract infection in hospitalized children under years of age in two main paediatric hospitals in hcmc ( - ) and the antibiotic use in out-patients with acute respiratory infections in children's hospital in hcmc ( - ). respiratory swabs were taken from all enrolled children. patients positive for generic ev rt-pcrs in csf or swabs from these studies and with diagnostic specimens available were included in this study. the geographical distribution of included patients is shown in additional file : figure s . viral rna was extracted from clinical samples using either the magna pure platform (roche applied science, darmstadt, germany) or the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany) following the manufacturer's instructions. ev-a specific one-step real-time rt-pcr ev-a detection and typing of samples from patients with cns infection was performed using real-time rt-pcr as described previously [ ] . in brief, μl of viral rna were subjected to one-step real-time rt-pcr reaction using the superscript iii one-step qrt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca, usa). the cycling conditions included one cycle of °c for min, followed by min at °c and min at °c, and cycles of s at °c, min at °c (including data acquisition) and s at °c. nested rt-pcr for viral protein (vp ) and complete viral protein and partial viral protein (vp /vp ) was carried out using previously described assays [ ] [ ] [ ] . vp rt-pcr of samples from patients with cns infection was primarily performed using the assay described by leitch et al., [ ] and alternatively, the vp assay described by oberste et al., [ ] . the vp /vp assay described by mirand et al., [ ] was applied to all samples from patients with respiratory infection and to samples from patients with cns infection with negative vp rt-pcrs. there was no major modification of the original assays except the use of superscript iii reverse transcriptase with platinum taq (invitrogen) in the first round rt-pcr and hotstar taq dna polymerase (qiagen) in the second round pcr and the adaptation of thermal cycling conditions as per supplier's instructions. in the three assays, μl of viral rna were used in the first round one-step rt-pcr and then . μl of the first round pcr product was transferred to the second round pcr reaction. pcr products of the nd round pcr were analysed in agarose gel, purified with cold ethanol and were then subjected to dna sequencing using the big dye terminator v . cycle sequencing kit (applied biosystems inc., foster city, ca, usa). nucleotide sequences of the vp and vp /vp were assembled using vector nti ® express software v . (thermo fisher scientific, waltham, usa). all the sequences have been submitted to genbank (mh -mh ). multiple sequence alignments were performed using bioedit software v . . (ibis therapeutics, ca, usa) and with the inclusion of reference prototype sequences obtained from genbank. neighbour-joining trees were constructed in mega software v . . (www. megasoftware.net) using the maximum composite likelihood nucleotide substitution model with bootstrap replicates. serotype assignments for vp and vp /vp sequences were performed using an automated phylogenetic-based enterovirus typing tool developed by kroneman et al., [ ] available at http://www.rivm.nl/mpf/enterovirus/ typingtool/. because the automated typing tool can determine vp /vp sequences at species level only [ ] , further serotype determination for vp /vp sequences was based on highest nucleotide identity score (hnis) and highest amino acid sequence similarity (haass) with reference prototypes [ , ] . in case of discrepancy, final assignment was based on haass and further confirmed using blast [ , ] . statistical analyses were performed using spss v . (spss, inc., chicago, il, usa). categorical variables were compared using chi-square or fisher's exact tests and continuous variables were compared using mann-whitney u test. two-sided p values ≤ . were considered significant. samples from a total of patients were included in this study, including from patients with cns infection and with respiratory infection. when analyzing for the monthly distribution of cases, there was no clear peak among cns cases, whereas two peaks of respiratory cases were found in april and november (fig. ). among patients with cns infection (n = ), were children (median age years, interquartile range [irq]: - ) and were adults ( years, irq: - . ). all adults had ev detected in csf and % ( / ) had meningitis as discharge diagnosis. of the children, had ev detected in csf, and the remaining ( / ) had ev detected in respiratory and/or rectal swabs. encephalitis was the most common discharge diagnosis ( / ). fifteen deaths were noted, and all were children with ev detected in swabs only (table ) . among patients with respiratory infections, the median age was . months (iqr: . - . ). inpatients (n = ) were younger than outpatients (n = ), due to study enrolment criteria ( table ) . bronchiolitis (as assessed at the discretion of treating physicians) was the most common clinical diagnosis ( % in outpatients and % in inpatients). thirty-five percent of inpatients had a clinical diagnosis of pneumonia ( table ) . in patients with cns infection, co-infection was more common and was found in % ( / ) children versus % ( / ) adults (table ) . most co-infected children ( / ) had ev detected in swabs only. of the documented fatal cases, were serologically confirmed to have co-infection with dengue virus (denv; n = ) and japanese encephalitis virus (jev; n = ) (additional file : table s ). among the co-infected adults, had cytomegalovirus (cmv) dna detected in csf and had positive csf culture with mycobacterium tuberculosis (table ) . in both patient groups of respiratory infection, there were high rates of co-infection with other respiratory viruses. co-infection with rhinovirus (rhv; %) and respiratory syncytial virus (rsv; %) was more common than other viruses, including adenovirus (adv; %), parainfluenza viruses (piv; %), human metapneumovirus (hmpv; %), human bocavirus (bov; %), human coronavirus (hcov; %), influenza virus (flu; %), and parechovirus (pev; %). the rate of coinfection with rhv and rsv was significantly higher in the inpatient group as compared to the outpatient group (p < . ) ( table ) . additional file : figure s ) and vp /vp (n = ; additional file : figure s ) sequencing. ev b was the only species detected in adults and the most common species in both swabs and csf from children (table ) . ev-a was the most commonly detected single serotype, in swabs and csf of children. three of these children had a clinical diagnosis of hfmd while the other children had a clinical diagnosis of encephalitis (additional file : table s ). there was no specific serotype associated with fatal outcome (additional file : table s ). among patients with respiratory disease different serotypes of four enterovirus species were identified by vp / vp sequence analysis including a (n = , serotypes), b other discharge diagnoses included sepsis (n = ), dengue haemorrhagic fever (n = ), colitis (n = ), diarrhea (n = ) metabolic disease (n = ), and hepatitis (n = ) c statistical comparison between children and adults with ev detection in csf (n = , serotypes), c (n = , serotypes), and d (n = , serotype). cv-a (n = ) and ev-d (n = ) were the two most common serotypes detected (table ) and these viruses had a close genetic relationship in phylogenetic tree analyses (additional file : figure s ). there is limited information on circulating enterovirus serotypes and associated clinical phenotypes from the asia pacific region, including viet nam. such knowledge is essential for laboratory diagnostics, patient management and future outbreak response. here, the diversity of enterovirus serotypes belonging ev a-d was assessed in samples from patients with cns and respiratory infections. some of these serotypes, including ev-a and ev-d have recently been recognized as emerging viral pathogens with pandemic potential. ev-a is the most important pathogen of hfmd epidemics in the asia-pacific region with over million cases reported annually [ , ] . here, ev-a was detected in / ( swabs and csf) children with cns infections, three of those had a clinical diagnosis of hfmd of which two died [ ] . in / cases, ev-a was the only pathogen detected in diagnostic samples. these data are in accordance with previous reports showing that ev-a has the potential to cause brain stem encephalitis and death in children under years of age [ ] , but is rarely detected in csf [ ] . ev-d has recently been recognized as an important cause of respiratory illness [ ] . ev-d emergence was first reported from two children's hospitals in the usa in august [ ] and, shortly after, from countries across the americas, europe, and asia [ ] . here, ev-d accounted for / successfully typed enteroviruses from patients with respiratory illness recruited between and . it remains unknown why ev-d has been circulating in viet nam for some time but has not been associated with outbreaks as observed in the usa and other countries in southeast asia. we have recently reported an in depth phylogenetic analysis of these ev-d viruses showing that their introduction into viet nam may have already occurred in [ ] . cv-a has been associated with herpangina, acute flaccid paralysis and epidemics of acute haemorrhagic conjunctivitis [ ] . in the present study, it was one of the most common enteroviruses: / typed viruses from patients with respiratory illness. however, the circulation of this particular enterovirus serotype has not been reported from viet nam. ev b was found in csf of both children and adults and / children and / adults had a clinical diagnosis of meningitis (additional file : table s ), in accordance with the known clinical spectrum of ev b [ , ] . ev a was found in csf of children and both had a clinical syndrome of encephalitis (additional file : table s ). it is likely these children had hfmd associated rhombencephalitis, in line with reports from the region starting in [ ] . remarkably, none of the children who died had either ev or any other pathogen detected in csf suggesting other etiology and/or emphasizing the diagnostic challenges of encephalitis [ , ] . in addition to ev-a , other common serotypes in cns infections cases included e- and e- (both ev b). these two viruses have frequently been involved in outbreaks of aseptic meningitis worldwide [ , ] . while e- has been confirmed to be the cause of a meningoencephalitis outbreak in northern viet nam previously [ ] , e- has yet been associated with any local epidemic of viral meningitis among children and young adults. pv- and (ev c) were detected in swab specimens of children: one with cns and two with respiratory illness. as children were under and sequences had - % of vp /vp identity to sabin vaccine strains [ ] these were considered to be derived from oral polio vaccine. data from this study showed that among patients with cns infection, children had a higher rate of co-infection than adults. here, this was due to the fact that both swabs and csf were used in the diagnostics for children while only csf was used for adults. in addition, children may be more vulnerable to viral infection than adults, particularly to the locally endemic viruses jev (n = ) and denv (n = ) [ ] . as jev and denv infections are commonly associated with severe disease and mortality, the contribution of ev to the disease phenotype of these cases is uncertain. similarly, in patients with respiratory infection, the clinical significance of ev infection should be interpreted with caution because of the high rate of co-infection with more common respiratory viruses [ ] . all the serotyping assays used in the current study, including the ev-a specific rt-pcr, vp and vp / vp sequencing have previously shown their efficient performance in clinical samples. the application of these assays in samples of patients with cns infection would help to improve serotyping yield. however, the overall serotyping success rate for patients with cns infection in the current study was low ( %) as compared to previous studies [ , ] . this may be due to low viral load or nucleic acid degradation after long-term storage. the low serotyping success rate ( %) of respiratory samples can be explained by the sole use of vp /vp sequencing assay. however, sample degradation and false positivity for ev using generic pcr among rv co-infected patients ( . %) could not be excluded. other drawbacks of the current study included the small sample size, the sporadic and expanded geographical distribution of cases that hampered our further analyses for serotypeassociated seasonality and geographics [ ] . despite these limitations, however, our study revealed diverse enterovirus serotypes have been circulating in viet nam in association with respiratory and cns infections. our study illustrates the circulation of diverse enterovirus serotypes belonging to four species (a-d), and their association with respiratory and cns infections in viet nam. these data are important for patient management, laboratory diagnostics and future outbreak response. additional file : figure s . the geographic distribution of cns and respiratory cases included in the current study (tiff kb) additional file : figure s . phylogenetic tree analysis of partial vp enteroviral sequences. a middle-point rooted tree of partial vp sequences ( bp) showing the genetic relationship among species and serotypes of the current study (filled triangles) and with reference prototypes. table s . demographic, clinical and diagnostic information of the death cases. table s . demographic, clinical and laboratory information of cases with ev-a infection. table s . vp /vp nucleotide and amino acid similarity scores. epidemiology of meningitis and encephalitis in the united states from - the impact of increased molecular diagnostics hand, foot, and mouth disease in china, - : an epidemiological study surveillance for enterovirus d in colorado children reveals continued circulation an enhanced enterovirus surveillance system allows identification and characterization of rare and emerging respiratory enteroviruses in denmark centers for disease c, prevention. enterovirus surveillance-united states identification of enteroviral infection among infants and children admitted to hospital with acute gastroentritis in ho chi minh city, vietnam enterovirus infections of the central nervous system in children: an update enteroviruses in the early st century: new manifestations and challenges seven-fold increase in viral meningo-encephalitis reports in england and wales during - validation and utilization of an internally controlled multiplex real-time rt-pcr assay for simultaneous detection of enteroviruses and enterovirus a associated with hand foot and mouth disease epidemiologic and virologic investigation of hand, foot, and mouth disease, southern vietnam viral etiology of encephalitis in children in southern vietnam: results of a one-year prospective descriptive study viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city aetiologies of central nervous system infection in viet nam: a prospective provincial hospital-based descriptive surveillance study respiratory syncytial virus and other viral infections among children under two years old in southern vietnam - : clinical characteristics and disease severity viral aetiology of central nervous system infections in adults admitted to a tertiary referral hospital in southern vietnam over years outpatient antibiotic use in acute respiratory infections in ho chi minh city, vietnam. oxford university clinical research unit improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing prospective identification of enteroviruses involved in meningitis in through direct genotyping in cerebrospinal fluid direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the vp region an automated genotyping tool for enteroviruses and noroviruses a comparison of the vp , vp , and vp regions for molecular typing of human enteroviruses molecular identification of new enterovirus types, ev - , ev , and ev - , members of the species human enterovirus b basic local alignment search tool virology, epidemiology, pathogenesis, and control of enterovirus epidemiological and clinical characteristics of children who died from hand, foot and mouth disease in vietnam enterovirus infection in children with hand, foot, and mouth disease in shanghai, china: epidemiology, clinical feature and diagnosis global emergence of enterovirus d : a systematic review severe respiratory illness associated with enterovirus d -missouri and illinois enterovirus d in viet nam two outbreaks of acute hemorrhagic conjunctivitis in africa due to genotype iii coxsackievirus a variant variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus predominance of enterovirus b and echovirus as cause of viral meningitis in a uk population a new variant of echovirus associated with a large outbreak of aseptic meningitis an approach for differentiating echovirus and japanese encephalitis virus infections in acute meningitis/encephalitis: a retrospective study of cases in vietnam vaccinederived polioviruses and the endgame strategy for global polio eradication detection of respiratory viruses in gargle specimens of healthy children we are indebted to ms. le kim thanh for providing supports in sample storage. the research leading to these results has received funding from the wellcome trust of great britain [ /z/ /z and /z/ /z]. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. le van tan is a wellcome-trust intermediate fellow in public health and tropical medicine. all data generated or analysed during this study are included in this published article. hrvd, lvt and ttt: conceived and designed the study. nttcb, nnn, lvt and ttt: did laboratory testing and analysed the test results. nnqm, ptq, tthc, hdtn, lahd, nvvc and gt: provided research materials, enrolled patients and took samples. nttcb, lvt, hrvd and ttt: drafted and reviewed earlier versions of the manuscript. all authors read and approved the final manuscript. the original studies were reviewed and approved by the local institutional review boards of all enrolling hospitals and the oxford tropical research ethics committee (oxtrec), university of oxford, oxford, united kingdom. written informed consent was obtained from parent or legal guardian of each participant. subsequent approvals for use of original samples for serotyping of enterovirus was obtained from the institutional review boards from the enrolling hospitals when not part of the original protocols. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -lh rhhe authors: loo, liat hui; jumat, muhammad raihan; fu, yi; ayi, teck choon; wong, pui san; tee, nancy ws; tan, boon huan; sugrue, richard j title: evidence for the interaction of the human metapneumovirus g and f proteins during virus-like particle formation date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: lh rhhe background: human metapneumovirus (hmpv) is now a major cause of lower respiratory infection in children. although primary isolation of hmpv has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious hmpv have hampered biochemical studies on the virus. these experimental methodologies usually require higher levels of biological material that can be achieved following hmpv infection. in this study we demonstrate that expression of the hmpv f, g and m proteins in mammalian cells leads to hmpv virus-like particles (vlp) formation. this experimental strategy will serve as a model system to allow the process of hmpv virus assembly to be examined. methods: the hmpv f, g and m proteins were expressed in mammalian cell lines. protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. vlp formation was examined using sucrose density gradient centrifugation and electron microscopy analysis. results: analysis of cells co-expressing the f, g and m proteins demonstrated that these proteins interacted. furthermore, in cells co-expression the three hmpv proteins the formation vlps was observed. image analysis revealed the vlps had a similar morphology to the filamentous virus morphology that we observed on hmpv-infected cells. the capacity of each protein to initiate vlp formation was examined using a vlp formation assay. individual expression of each virus protein showed that the g protein was able to form vlps in the absence of the other virus proteins. furthermore, co-expression of the g protein with either the m or f proteins facilitated their incorporation into the vlp fraction. conclusion: co-expression of the f, g and m proteins leads to the formation of vlps, and that incorporation of the f and m proteins into vlps is facilitated by their interaction with the g protein. our data suggests that the g protein plays a central role in vlp formation, and further suggests that the g protein may also play a role in the recruitment of the f and m proteins to sites of virus particle formation during hmpv infection. human metapneumovirus (hmpv) is a new member of paramyxoviridae that was first identified in children with respiratory diseases in netherlands [ ] . the clinical symptoms that are caused by hmpv infections in children are similar to those observed with respiratory syncytial virus (rsv) infection; ranging from mild symptoms to pneumonia. hmpv is now a globally recognised cause of lower respiratory infection in children [ , ] ). genetic analysis identified two major genogroups a and b [ ] [ ] [ ] . hmpv expresses two major integral membrane proteins that play a role in virus entry. the attachment (g) protein plays a role in virus attachment and is expressed as a single polypeptide chain, which subsequently undergoes extensive nand o-linked glycosylation [ ] . the fusion (f) protein mediates fusion of the virus and host-cell membranes, and is initially synthesised as a single polypeptide chain (f ) that undergoes proteolytic cleavage to generate the mature and active form of the protein, consisting of f and f protein subunits [ ] . the virus also expresses a third membrane-associated protein called the matrix (m) protein, which is analogous to the m protein of rsv and is a major determinant of virus morphology [ ] . primary isolation of hmpv has been achieved in several different cell lines [ , , ] , however tissue culture adapted isolates can require up to days incubation before cytopathic effects are visualised [ , [ ] [ ] [ ] . this low level of virus replication and the subsequent recovery of low levels of infectious hmpv in standard cell culture have hampered biochemical studies on the virus. these experimental methodologies usually require higher levels of biological material than can be achieved following hmpv infection. virus-like particle (vlp) formation following the coexpression of specific virus structural proteins has been demonstrated in several paramyxoviruses [ ] [ ] [ ] [ ] [ ] . these studies have allowed the identification of essential virus proteins that are required for virus particle assembly. although a central role for the m protein in vlp formation has been reported for human parainfluenza type virus [ ] and newcastle disease virus [ ] , the expression of the m protein alone was insufficient for vlp production in simian virus type [ ] and avian pneumovirus type c [ ] . the use of recombinant hmpv protein expression to drive the formation of similar hmpv vlps can potentially overcome the problems associated with the poor cultivation of hmpv in tissue culture. in addition, by direct cloning of the virus genes from clinical material the expression of gene sequences that have not been subjected to extensive tissue culture adaptation can be examined. this therefore affords a relatively simple experimental system with which to examine hmpv morphogenesis. in this study we have examined the capacity of the hmpv f, g and m proteins to form vlps in mammalian cells, and to further examine the minimal virus protein requirements that lead to vlp formation. expression of the hmpv f, m and g proteins in mammalian cells the hmpv f and g genes were cloned directly from hmpv strain sin -ntu and the m gene from sin -ntu [ ] without prior passage of the virus in tissue culture. a cmyc or flag tag epitope was placed on the c-terminus of the f and g proteins respectively to generate pcaggs/f-cmyc and pcaggs/g-flag respectively. the m gene was cloned without an epitope tag to generate pcaggs/m. the hmpv strain sin -ntu and sin -ntu were identified on the basis of sequence analysis as being of the a subtype. t cells transfected with either pcaggs, pcaggs/f and pcaggs/g were surface-biotinylated and immunoprecipitated using either anti-cmyc or anti-flag antibodies ( figure a ). the f protein migrated as a kda protein species (f ), which is the expected size for the uncleaved hmpv f protein [ , ] . an oligomeric f protein species was also detected ( figure b , highlighted by f ) which has also been reported previously [ , ] . the glycans attached to the f protein were resistant to endo-h-treatment, but pngaseftreatment resulted in the formation of a kda protein species (f ), the expected size for the deglycosylated f protein. tpck-trypsin treatment of the recombinant f protein yielded the presence of a protein species corresponding in size to the f protein subunit ( figure b) , in a similar manner to that described previously [ ] . collectively these data are consistent with the authentic expression of the f protein. in pcaggs/g-flag transfected cells the g protein migrated as two relatively broad endo-h-resistant protein bands of approximately kda (g ) and kda (g ) ( figure a ). the size of g was consistent with monomeric g protein, while the ga kda protein species was consistent with an oligomeric form of g protein. several studies have demonstrated the propensity of the hmpv g protein to form oligomers when analysed by immunoblotting. both g and g were endo-h resistant, however following pngasef treatment the g protein was reduced to a faint protein smear. this indicated removal of the n-linked glycans, and the residual protein smear was presumably due to heterogeneity of the remaining attached o-linked glycans [ ] . the glycosylation analysis was consistent with the presence of mature forms of the surface-expressed f and g proteins. the presence of unusually high levels g protein oligomers in sds page analysis has been reported previously in avian metapneumovirus [ , ] . due to its location beneath the plasma membrane, as expected we failed to detect the presence of biotinylated m protein. immunoblotting of lysates prepared from pcaggs/m transfected cells with mabm f (anti-m) revealed the presence of a kda protein, the expected size of the hmpv m protein ( figure c ). lysates prepared from pcaggs, pcaggs/g-flag or pcaggs/f-cmyc transfected cells were immunoprecipitated using either anti-cmyc or anti-flag and the immunoprecipitates then immunoblotted with either anti-cmyc ( figure d ) or anti-flag ( figure e ). only pcaggs/f-cmyc transfected cells immunoprecipitated and immunoblotted with anti-cmyc showed the presence of f proteins species corresponding in size to the monomeric and oligomeric f protein ( figure d ). in a similar analysis g was only detected in pcaggs/g-flag transfected cells immunoprecipitated and then immunoblotted with anti-flag ( figure e ). this demonstrated the specificity of the immunoprecipitation assay. trypsin degradation of the g protein has been previously reported [ ] , and we have also noted g protein degradation in the presence of quantities of trypsin that are required for f protein activation (jumat and sugrue, unpublished observations). therefore, in all subsequent studies involving g protein expression the trypsin treatment was omitted. cells were co-transfected with pcaggs/f-cmyc and pcaggs/g-flag, surface-biotinylated and immunoprecipitated with anti-flag. a small but discernible increase in the apparent size of the two g protein species from g to g and from g to g was noted ( figure a) . similarly, surfacebiotinylation of hmpv-infected cells and immunoprecipitated with anti-g showed the presence of g and g ( figure b ). this indicated modified processing of the g protein in co-transfected cells, resulting in an apparent molecular mass that is more similar to that observed in hmpv-infected cells. although this suggested an interaction with the f protein, immunoprecipitation of the co-transfected cells with anti-cmyc or anti-flag and then immunoblotted using anti-cmyc ( figure c ) or anti-flag ( figure d ) failed to demonstrate co-precipitation of these proteins. in a similar manner immunoprecipitation of the f protein from hmpv-infected cells showed the presence of biotinylated species corresponding in size to the f and f proteins ( figure b ). similar ratios of the f and f proteins were observed following trypsin treatment of the recombinant f protein expressed in t cells ( figure b) . the f and g proteins are both integral membranes and we hypothesized that the presence of the lipid membrane may be required for stabilization of the protein complex in situ. in this scenario the removal the lipid membrane by detergent extraction prior to immunoprecipitation could lead to destabilization of the protein complex. we therefore used dithiobis [succinimidylpropionate] (dsp) to stabilise the protein complex by in situ cross-linking prior to detergent extraction of the virus proteins as described previously [ , ] . although dsp cross-linked complexes were predicted to be beyond the resolution of sds-page analysis (> kda), following immunoprecipitation the individual components of the stabilised protein complexes could be released by removal of the covalent cross-links by reductive cleavage using β-mercaptoethanol-treatment. t cells were co-transfected with pcaggs/f-cmyc and pcaggs/g-flag, and the cell monolayers treated with increasing dsp concentrations. cell lysates were prepared and immunoprecipitated with anti-cmyc, and the presence of the g protein and f protein in the immunoprecipitation assays detected by immunoblotting with anti-flag ( figure e ) and anti-cmyc ( figure g ) respectively. immunoblotting with anti-flag revealed the appearance of increasing amounts of a kda flag-tagged protein (the expected mass of ga protein) with increasing dsp concentrations ( figure e ). this protein was not present in lysates prepared from non-treated cells. probing with anticmyc revealed similar levels of the monomeric (f ) and oligomeric (f ) f protein species in both non-treated and dsp-treated cell monolayers ( figure g ). similarly lysates immunoprecipitated with anti-flag and immunoblotted using anti-cmyc revealed increasing amounts of f with increasing dsp concentration, together with a cmyc-tagged protein species > kda ( figure f ) whose molecular mass was consistent with higher oligomeric forms of the f protein. probing with anti-flag revealed monomeric (g ) and oligomeric forms (g ) of the g protein in both non-treated and dsp-treated monolayers ( figure h ). these data suggested that dsp was able to stabilise a protein complex that formed between the f and g proteins. the sedimentation characteristic of cross-linked f and g protein complex was examined using sucrose this was clarified and loaded onto a - % sucrose gradient. the gradient was fractionated and each fraction (i) ip with anti-cmyc and ib with anti-flag, or (j) ip with anti-flag and ib with anti-cmyc. the various monomeric and oligomeric f and g protein species are indicated. high molecular mass f proteins are highlighted with "*". gradient centrifugation. cells were co-transfected with pcaggs/f-cmyc and pcaggs/g-flag and treated with . mm dsp. a detergent extract was prepared and then applied to a continuous - % (w/v) sucrose gradient. after centrifugation the gradient was fractionated, and the individual fractions immunoprecipitated with anti-cmyc and immunoblotted with anti-flag ( figure i ) or immunoprecipitated with anti-flag and immunoblotted with anti-cmyc ( figure j ). this assay revealed that the co-precipitating f and g proteins were mainly located within fractions to , consistent with the co-migration of the dsp stabilized f and g protein complex. we also used fluorescence scanning confocal microscopy (fscm) to examine the distribution of the g and f proteins in pcaggs/g-flag and pcaggs/f-cmyc ( figure ) co-transfected cells. in pcaggs/g-flag transfected cells stained using anti-flag a filamentous staining pattern was observed ( figure a ). pcaggs/fcmyc transfected cells stained using anti-cmyc exhibited a more punctate staining pattern ( figure b ). in cells cotransfected with pcaggs/g-flag and pcaggs/fcmyc and stained with anti-cmyc and anti-flag a high degree of co-localisation between the f and g proteins within small filamentous projections was observed ( figure c ). this was supported by statistical analysis using manders and pearsons correlation coefficients which showed a high level of co-localisation ( figure d and e). figure b ). in both cases co-precipitation of oligomeric forms of the f protein ( figure a ) and g protein ( figure b ) with the m protein was detected. these immunoprecipitation assays were immunoblotted using anti-m which confirmed the presence of the m protein ( figure c ). we failed to detect the presence of the m protein in detergent extracts prepared from pcaggs/m transfected cells that were immunoprecipitated with influenza virus anti-np (i.e. a non-specific antibody) and immunblotted with anti-m ( figure c ); confirming the specificity of the m protein immunoprecipitation. the fm-and fgm-transfected cells were immunoprecipitated with anti-cmyc ( figure d) , and the gm-and fgm-transfected cells immunoprecipitated with anti-flag ( figure e ). immunoblotting of the immunoprecipitation assays with anti-m revealed the presence of the m protein in each transfection combination. the presence of hmpv proteins in pc-transfected cells were not detected using either antibody. collectively, these data provided evidence of an interaction between the m protein and the virus glycoproteins. co-expression of the f, g and m proteins lead to the formation of virus-like particles we hypothesized that the interaction of the hmpv g, f and m proteins could lead to the formation of structures that resembled hmpv particles. these structures would be expected to be cell membrane-bound, and once detached from the cell, they would be expected to have a corresponding buoyant density that could be assessed using density gradient centrifugation. the isolation of hmpv vlps was achieved based on a modified method for the purification of the related rsv [ ] . cells were transfected with pcaggs (mock-transfected) or cotransfected with pcaggs/f-cmyc, pcaggs/g-flag and pcaggs/m (fgm-transfected) and at h posttransfection the cells were harvested and processed for vlp isolation as described in methods. the clarified cell preparations were loaded onto % w/v sucrose cushions, and after centrifugation the resulting pellet subsequently resuspended and loaded onto a discontinuous step sucrose gradient consisting of %, % and % sucrose (w/v) concentrations. after centrifugation the material at the - % (w/v), - % (w/v) and - % (w/v) sucrose interfaces (additional file : figure s ) was harvested and examined by immunoblotting using anticmyc ( figure a ), anti-flag ( figure b ) and anti-m ( figure c ). all three hmpv proteins were found to be present at the - % sucrose density interface in fgmtransfected cells, while no hmpv proteins were detected in the discontinuous step sucrose gradient prepared using the mock-transfected cells ( figure d , e, f). in a similar analysis the resuspended pellet recovered from the % sucrose cushion was applied to a continuous % to %(w/v) sucrose gradient. after centrifugation the gradient was fractionated, and the individual fractions proteins examined by immunoblotting using anti-cmyc ( figure a ), anti-flag ( figure b ) and anti-m ( figure c) . the f and g proteins were primarily detected in fractions to , and this co-migration was consistent with the identification of the protein complex identified above. we noted that while the m protein appeared to co-migrate with the f and g proteins in the continuous gradient, the m protein peak fraction exhibited a slight shift (by fraction), suggesting that the m protein may be associated with more than one type of membrane-bound buoyant particle. analysis of mock-transfected and fgm-transfected cells was performed by scanning electron microscopy (sem). cells were imaged using conventional sem and were examined at relatively low magnification. this showed that while mock-transfected cells exhibited a relatively smooth surface morphology at this magnification, fgm-transfected cells showed an increase in a filamentous morphology ( figure a ). mock-transfected cells and fgm-transfected cells were examined in more detail using field emission sem (fesem) as described previously [ ] . cells were stained using anti-flag and anti-mouse igg conjugated to nm colloidal gold, and the surface morphology and distribution of gold label (indicating g protein distribution) examined. while unlabelled surface features corresponding to microvilli could be detected on the surface of mock-transfected cells, labelled filamentous projections were detected on the surface of fgm-transfected cells ( figure b and c) . this was similar in appearance to the filamentous g protein staining pattern exhibited when hmpv-infected cells were stained using anti-g and examined by fscm ( figure d) . collectively, the sedimentation centrifugation and imaging analysis was consistent with the formation of filamentous structures on fgm-transfected cells, which were similar in appearance to the filamentous structures detected on hmpv-infected cells. the capacity of the individual hmpv proteins to form vlps was examined using the discontinuous density gradient centrifugation vlp assay described above. in pcaggs/ g-flag transfected cells the g protein was detected in the total cell extract, confirming expression ( figure a ). in the vlp assay immunoblotting with anti-flag revealed g protein species of sizes kda (g ) and kda (g ) exclusively in the - % (w/v) sucrose interface. in contrast, although f protein expression was detected in pcaggs/f-cmyc transfected cells, the f protein was not detected in either of the three fractions ( figure b ) suggesting that it was not efficiently incorporated into the vlp fraction. similar levels of the m protein were detected in the - % (w/v) and - % (w/v) sucrose interfaces ( figure c ), suggesting that the m protein was associated with membrane-bound structures with at least two different buoyant densities. a similar analysis was performed on fg-transfected, gm-transfected, and fm-transfected cells. in fgtransfected cells the presence of the f and g proteins was detected in the cell extract confirming expression ( figure d ). however, in fg-transfected cells f (see figure on previous page.) figure hep- cells co-expressing f and g proteins showing co-localisation of f and g proteins. cells expressing either (a) the g protein or (b) the f protein were stained using anti-flag and anti-cmyc respectively and imaged using confocal microscopy at an optical plane corresponding to the cell periphery and cell top. (c) cells co-expressing the f and g proteins were stained using anti-flag and anti-cmyc and imaged using confocal microscopy at an optical plane corresponding to the cell top and cell periphery. (d) co-localisation was calculated using regions (white circles) within the co-transfected cells. wcc( )-weighted co-localisation coefficient (green channel), wcc( )-weighted co-localisation coefficient (red channel), mand-manders overlap coefficient, r pearson's correlation coefficients. (e) an example of the scatter-plot region from one of these circles is shown. the x-axis ( ) denotes green pixel distribution, the y-axis ( ) denotes red pixel distribution and region denotes the yellow pixel combination (co-localising pixels). together with g and g was detected at the - % (w/v) sucrose interface. this indicated that co-expression of the f and g proteins facilitated incorporation of f into the vlp fraction. in gm-transfected cells the presence of the m and g proteins were detected in the cell extract confirming expression ( figure e ). however, a significantly higher level of m protein was detected in the - % (w/v) sucrose interface together with the two g protein species. interestingly, although both proteins were present within the - % (w/v) sucrose interface we noted no change in the electrophoretic migration of the g protein. in fm-transfected cells the presence of the f and m proteins were detected in the cell extracts ( figure f ), however the levels of f and m protein in the sucrose interfaces were similar to those detected in cells singly expressing either the f protein ( figure b ) or m protein ( figure c ). the hmpv f and g proteins have also been shown to bind heparan sulphate [ ] and heparin [ ] respectively. the hmpv g protein exhibits many similarities to the corresponding rsv g protein e.g. it is heavily modified by o-linked glycosylation and can bind glycosaminoglycans [ , , ] , which suggests a similar role for the hmpv g protein during hmpv infection. evidence for the interaction between the rsv f and g proteins in the virus envelope [ , ] has provided support for a single protein complex involving both proteins. in this present study we have also provided evidence for the existence of a similar protein complex involving the hmpv f and g proteins. by analogy with rsv, we can hypothesise that such a protein complex will form during hmpv replication. our study indicated that co-expression of the hmpv protein was able to form vlps, and the imaging analysis indicated that the recombinant hmpv particles formed as filamentous structures in a similar manner to that in hmpv-infected cells. similar virus filaments are formed during morphogenesis of the closely related rsv, suggesting some common features in the assembly process of these different viruses. in this regard we noted that the hmpv g protein facilitated vlp formation independently of other hmpv proteins. our data also indicated that the interaction of the g protein and the m and f proteins facilitated the incorporation of the latter into these structures. although the m protein has been proposed to play a role in rsv morphogenesis, recent data suggests that it does not play a role in the initiation of virus particle formation [ ] . our data suggested that expression of the g protein in the absence of other virus proteins is sufficient to form these structures, and its interaction with other virus (and possibly other unknown cell factors) may lead to their active recruitment into vlps. in particular, our data suggested the presence of the f protein in these structures is largely dependent on its association with the g protein. it is likely that there are significant differences in the homeostasis in virus-infected and transfected cells which can influence virus processes. although the vlps and virus particles are similar in appearance it is also likely that there will be subtle differences between the virus architecture in vlps and mature virus particles that formed on infected cells [ ] . however, given these caveats, we noted an overall similar morphology in the hmpv vlps and hmpv particles. given the technical difficulties in growing hmpv in mammalian tissue culture cells (in particular low passage clinical isolates), the generation of hmpv vlps in tissue culture cells may form the basis of an experimental system to examine aspects of the hmpv maturation process, in particular using protein sequences derived from non-tissue culture adapted viruses). these vlps may also be a potential source of particulate virus antigen that can form the basis of a vaccine candidate, and future studies will examine these possibilities. cloning of the hmpv f, g and m genes from nasopharyngeal washings nasopharyngeal washing were collected from children admitted to kk women's and children's hospital for respiratory infection as described previously [ ] . the f, g and m genes were amplified from the hmpv a positive nasopharyngeal washings (sin -ntu ) using the primers f pcaggf f pcaggmycr, g pcaggf g pcaggflagr, m pcaggf and m pcaggr (additional file : table s ). the pcr products were then inserted into the vector pcaggs [ ] . the anti-flag and anti-cmyc were purchased from sigma aldrich and cell signalling respectively. m f (anti-m) was prepared from bacterially expressed hmpv m protein. the hmpv g (at ) and f proteins (mab ) were obtained from geoff toms [ ] . the hmpv a stain ncl - / was used in the llc-mk cell line in dmem (bsa, . μgml tpck-trypsin) at °c for days. cells were transfected using lipofectamine reagent (invitrogen, usa) following the manufacturer's instructions. the media was changed after hr and the cells were incubated at °c for hr before further analysis. the proteins were separated by sds-page, transferred onto pvdf membranes (immobilon-p, milipore, usa) as described previously. the tagged proteins were detected with rabbit anti-flag antibodies (sigma-aldrich, usa), mouse anti-cmyc antibodies (cell signaling technology, usa) or mouse anti-m antibodies (nanyang technological university) as appropriate. protein bands were visualised using the ecl system (ge healthcare, usa). molecular masses were estimated using kaleidoscope markers (biorad, usa). cell extracts were prepared at °c using rip buffer ( % np- , . % sds, mm nacl, mm edta, mm pmsf, mm lysine, mm tris-hcl, ph . ) and clarified by centrifugation ( , g, min °c) and immunoprecipitated as described previously [ ] using appropriate antibodies. the immunoprecipitation assays were separated using sds-page. cells were surface-labelled using ez-link sulfo-nhs-lc-lc-biotin (pierce biotechnology, usa) as described previously [ ] . briefly, cell monolayers were incubated in . mg/ml solution of ez-link sulfo-nhs-lc-lc-biotin (pierce biotechnology, usa) in pbs ph for hr at room temperature. the lysates were immunoprecipitated with either anti-cymc or anti-flag as appropriate. in situ cross-linking was performed using dithiobis [succinimidylpropionate] (dsp) (pierce biotechnology, usa) as described previously [ ] . briefly, the cell monolayers were treated with between . and . mm dsp ( mm stock solution in dmso) in pbs ph . , and the cells were incubated at room temperature for hour. the monolayers were then washed with pbs ph . containing mm lysine prior to detergent extraction with rip buffer. transfected cells were lysed at °c for min in denaturation buffer ( · % sds, mm dtt). the samples were then made up to a final concentration of either mm sodium phosphate + % np- (ph ) or mm sodium citrate (ph ) and incubated at °c overnight with u pngase f (new england biolabs, usa) or u endo _ h (new england biolabs, usa) respectively. all steps were performed at °c. cell monolayers were extracted using rip buffer, and the resulting lysate clarified by centrifugation ( , g, min). the clarified lysate was layered onto a - % sucrose (w/v) gradient prepared in ten buffer ( mm edta, mm nacl, mm tris-cl ph + . % triton x- ). the gradient was centrifuged for hr at , g and °c (hitachi cp wx preparative ultracentrifuge; hitachi co ltd, japan). cell suspension was subjected to freeze-thaw by ethanoldry ice and a °c water bath. after rounds of freezethaw, the cell suspension was clarified ( , g for min) and loaded onto a sucrose cushion ( % w/v sucrose in ten buffer), and centrifuged at , g for hr at °c (hitachi cp wx ultracentrifuge). the resulting pellet was resuspended in μl of ten buffer and loaded onto a discontinuous sucrose gradient ( %, % and % sucrose (w/v) in ten buffer). the material was harvested from each sucrose interface was harvested for further analysis. for the continuous centrifugation analysis the sucrose pellet harvested from the sucrose cushion was resuspended in ten buffer and applied to a continuous gradient ( % to % sucrose (w/v) in ten buffer) and centrifuged at , g for hr at °c. the gradient was harvested by removing ml fractions, which were then analysed further. cells transfected overnight on mm glass coverslips were fixed with methanol:acetone ( : ) for min at °c. the cells were labelled using anti-cmyc, anti-flag or anti-m antibodies and the appropriate secondary antibodies (conjugated to either fitc or alexa fluor ) as described previously [ ] . the cells were visualized using a zeiss axioplan confocal microscope using appropriate machine settings scanning electron microscopy (sem) cells were processed as described previously [ ] . briefly, transfected cells were fixed using . % glutaraldehyde and labeled using anti-flag and anti-rabbit igg ( / dilution) conjugated with colloidal gold ( nm) (sigma-aldrich, usa) for hr at room temperature. the cells were then post-fixed in . % glutaraldehyde and % oso prior to critical point drying. the cells were carbon coated and viewed using either a jeol or a jeol fe-sem using appropriate machine settings. osterhaus ad: a newly discovered human pneumovirus isolated from young children with respiratory tract disease epidemiology of human metapneumovirus the emergence of human metapneumovirus genetic diversity between human metapneumovirus subgroups analysis of the genomic sequence of a human metapneumovirus antigenic and genetic variability of human metapneumoviruses intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein characterization of human metapneumovirus f protein-promoted membrane fusion: critical roles for proteolytic processing and low ph paramyxovirus assembly and budding: building particles that transmit infections human metapneumovirus detection in patients with severe acute respiratory syndrome analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time pcr human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation human metapneumovirus m - protein inhibits viral transcription and replication human parainfluenza virus type matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures requirements for budding of paramyxovirus simian virus virus-like particles requirements for the assembly and release of newcastle disease virus-like particles quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein the cellular endosomal sorting complex required for transport pathway is not involved in avian metapneumovirus budding in a virus-like-particle expression system human metapneumovirus in children modification of the trypsin-dependent cleavage activation site of the human metapneumovirus fusion protein to be trypsin independent does not increase replication or spread in rodents or nonhuman primates an s p substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters pneumovirus-like characteristics of the mrna and proteins of turkey rhinotracheitis virus avian metapneumovirus sh gene end and g protein mutations influence the level of protection of live-vaccine candidates recombinant human metapneumovirus lacking the small hydrophobic sh and/or attachment g glycoprotein: deletion of g yields a promising vaccine candidate the rsv f and g glycoproteins interact to form a complex on the surface of infected cells the small hydrophobic (sh) protein accumulates within lipid-raft structures of the golgi complex during respiratory syncytial virus infection protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein in virus particle assembly distribution of the attachment (g) glycoprotein and gm within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy human metapneumovirus (hmpv) binding and infection are mediated by interactions between the hmpv fusion protein and heparan sulfate role of cellular glycosaminoglycans and charged regions of viral g protein in human metapneumovirus infection oligomerization and post-translational processing of glycoprotein g of human respiratory syncytial virus: altered o-glycosylation in the presence of brefeldin a contribution of the respiratory syncytial virus g glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo human respiratory syncytial virus surface glycoproteins f, g and sh form an oligomeric complex the human respiratory syncytial virus matrix protein is required for maturation of viral filaments influenza virus assembly and budding diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the north-east of england respiratory syncytial virus assembly occurs in gm -rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin- evidence for the interaction of the human metapneumovirus g and f proteins during virus-like particle formation we acknowledge the national medical research council, singapore, for research funding (nmrc/ / ). we thank geoff toms for the hmpv a stain ncl - / and the at (anti-g) and mab antibodies. l.h. loo was a recipient of a nmrc-lee foundation phd scholarship. additional file : figure s . discontinuous sucrose gradient concentration of virus-like particles from t cells. cells were processed for discontinuous density gradient centrifugation as described in text. after centrifugation the opalescent bands at each interface in the sucrose gradient was detected using a focused light. image of ultracentrifuge tube shows the presence of opalescent bands at the ( ) - % (w/v) sucrose, ( ) - % (w/v) sucrose and ( ) - % (w/v) sucrose interfaces in the analysis from pcaggs/f-cmyc, pcaggs/g-flag, pcaggs/m transfected cells is shown. the opalescent band at interface is highlighted (black arrow).additional file : table s . primers used for cloning of f, g and m genes into pcaggs vector. the underlined bases indicate restriction enzyme recognition sites. the bases in italics are those coding for cmyc or flag tags. the genbank/embl/ddbj accession numbers for the genome sequences of hmpv isolates sin -ntu f and g genes and sin -ntu m gene are ef , jq , jq respectively. the authors declare that they have no competing interests. key: cord- -vt yio x authors: pan, yongfei; tian, xiaoyan; li, wei; zhou, qingfeng; wang, dongdong; bi, yingzuo; chen, feng; song, yanhua title: isolation and characterization of a variant porcine epidemic diarrhea virus in china date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vt yio x an outbreak of diarrhea in pigs started in guangdong, south china in january . cases were characterized by watery diarrhea, dehydration and vomiting, with – % morbidity and – % mortality in suckling piglets. the causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (pedv). in this study, we isolated a pedv strain designated chgd- from piglet intestines using vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. the complete genome of chgd- was shown to be , nucleotides in length, with a similar structure to that of pedv reference strains. phylogenetic analyses based on the whole genome revealed that chgd- shared nucleotide sequence identities of . – . % with two other chinese isolates reported in the same year, thus constituting a new cluster. amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of chgd- and the field strain knu in korea. its orf and nucleoprotein genes, however, were divergent from all other sequenced pedv isolate clusters and therefore formed a new group, suggesting a new variant pedv isolate in china. further studies will be required to determine the immunogenicity and pathogenicity of this new variant. porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea (ped), an enteric disease characterized by vomiting, watery diarrhea, and dehydration in swine. this disease was first reported in feeder and grower pigs in the uk in [ ] , after which the virus was identified [ , ] . the disease has subsequently been reported in a number of european countries [ , ] , and more recently in china, korea, japan, thailand and vietnam [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pedv is an enveloped rna virus belonging to group a, genus coronavirus, family coronaviridae, within the order nidovirales. the viral genome is a single-stranded positive-sense rna of approximately kb in size, containing six genes: the replicase (rep), spike (s), orf , envelope (e), membrane (m), and nucleoprotein (n) genes, arranged in the order '-rep-s-orf -e-m-n- ' [ ] [ ] [ ] . as a coronavirus, pedv comprises three corresponding major viral structural proteins: the s ( - kda), m ( ) ( ) ( ) ( ) ( ) ( ) , and n ( - kda) proteins [ , ] . the s protein plays a pivotal role in determining viral-cellular fusion activity and in inducing an immune response in the natural host [ ] [ ] [ ] . the m protein plays an important role in the virus-assembly process, and induces antibodies that neutralize virus in the presence of complement [ ] [ ] [ ] . the n protein of coronaviruses forms a helical ribonucleoprotein with the virus genomic rna and is the predominant antigen produced in coronavirus-infected cells, thus making it a major viral target [ , ] . unlike the structural proteins, little is known about the functions of the accessory proteins. the recently-identified orf gene has been demonstrated to be a potentially important determinant of virulence in this virus [ , ] . pedv can generally be controlled using the vaccine strategy, and vaccination with killed or attenuated pedv vaccine has been widely carried out in china, where ped usually manifests a mild and enzootic pattern (lower mortality in suckling piglets). however, a severe acute diarrhea outbreak associated with high morbidity ( - %) and mortality ( - %) was observed in suckling piglets at approximately premises in guangdong, china, in early . although most sow herds had previously been vaccinated with both killed and attenuated pedv vaccines based on cv , some of these were still infected, showing transient diarrhea and anorexia, but not death. pedv field isolates thus need to be isolated and their molecular epidemiology investigated in order to better control and prevent future pedv outbreaks. in this study, a pedv strain was isolated from sick piglets during this outbreak and grown in vero cells. molecular characterization of the virus identified it as a variant pedv emerging in china. for pcr/rt-pcr detection of viruses, intestinal and fecal samples from sick piglets were first examined for the presence of tgev, porcine rotavirus, porcine reproductive and respiratory syndrome virus (prrsv), pcv , porcine kobuvirus and pbov. all assays were negative except for three pcv -positive samples. all specimens were subsequently examined for pedv, and all samples were positive (data not shown). all samples were tested for common pathogenic intestinal bacteria, such as salmonella and pathogenic escherichia coli, but no significant numbers of salmonella or e. coli were isolated from sick piglets from different premises. a distinct cpe was noted after seven passages in vero cells. the cpe was characterized by cell fusion, syncytia formation and eventual detachment from the plastic surface ( figure b) . the virus isolate designated chgd- was biologically cloned by three rounds of plaque purification in vero cells prior to further virus characterization. in addition, cells tested positive by ifa ( figure d ). electron microscopy of a negatively-stained sample revealed the presence of medium-sized viral particles of approximately - nm in diameter. in some of the virions, surface projections characteristic of coronaviruses were evident ( figure e ). phylogenetic analyses of the s protein amino acid sequences revealed that all pedv strains in this study could be separated into two groups: chgd- belonged to group , which also contained the two japanese isolates kawahira and nk, eight korean field strains (chinju , knu- , knu- , and knu- -knu- ) and two chinese strains (bj- - and ch/fjnd- / ), which were deposited in genbank in ( figure b ). the orf gene of chgd- was determined to be nucleotides in length, coding for a polypeptide of amino acids. the orf gene of chgd- shared . - . % amino acid identity with other pedv strains, and the highest identity with the korean virulent dr . based on phylogenetic analyses of the orf amino acid sequences, all the pedv strains could be divided into four groups (figure c ). group comprised cv , br / , sm and lzc strains; group consisted of vaccine strains (attenuated dr ); group was made up of eight korean field strains and chinese strains. these results showed some similarities with the previous report by park et al. [ ] . interestingly, phylogenetic analysis showed that chgd- formed a fourth group, clearly different from other pedv isolates. each group had unique differences in its sequences. group had a -amino acid deletion (at positions - ), which is a marker of attenuated vaccine. the m protein of chgd- was found to be amino acids long and highly conserved. it shared . - . % amino acid identity with the other pedv strains. the highest amino acid identity ( . %) was with the vietnamese field isolates (vn m and vn m ), which were isolated from southern vietnam during the - ped outbreaks. phylogenetic analyses demonstrated that all pedv strains were similarly divided into two distinct genetic groups ( figure d the n gene of chgd- was nucleotides in length, coding for a polypeptide of amino acids. the amino acid sequence had . - . % identity with other pedv strains, and the highest identity ( . %) with the ch/s and vaccine strains (attenuated dr ). alignment analysis indicated that the entire nucleocapsid protein of chgd- was generally highly conserved, but had amino acid substitutions compared to cv . it was predicted to contain eight potential serine (s)-linked phosphorylation sites and six potential threonine (t)-linked phosphorylation sites, including two protein kinase c phosphorylation sites, one casein kinase ii phosphorylation site, and one camp-and three cgmp-dependent protein kinase phosphorylation sites. based on analyses of the amino acid sequences of the pedv n proteins, all pedv strains could be divided into four groups (figure e ). chgd- differed from all other pedv isolates, and formed a new separate group. there were unique changes in the deduced amino acid sequences among the groups. two amino acid changes ( a t , v m ) were found in group ; k r , n s and q l occurred only in group ; groups , shared two specific amino acid changes ( s t , h l ); and g a was found in groups , and . noticeably, six amino acid mutations ( a s , n s , k l , a t , i v , q p ) were observed only in chgd- (figure c ). the clinical signs from the outbreak in guangdong, along with both macroscopic and microscopic examinations of the lesions, were strongly suggestive of pedv or tgev infection. tgev and other possible pathogens were excluded and pedv was therefore focused on as the only significant causative agent, followed by attempts to isolate it from piglets with the disease. pedv was isolated successfully and propagated in vero cell cultures in the presence of trypsin, according to the method described by hofmann and wyler, and by kusanagi et al. [ , ] . however, the cpe caused by the pedv isolate was not detected in vero cells until after seven passages, despite being detectable by pcr. this was in contrast to the results of hofmann and wyler, who observed a cpe even at passage one in the same cells, and a prominent effect from virus passage five [ ] . kusanagi et al. reported that a characteristic cpe was detected at passage two, and became more evident with subsequent passages of vero cells [ ] . this discrepancy may be attributable to the relative susceptibilities of vero cells to different isolates of pedv. to characterize the virus isolate, we determined the complete genomic sequence of chgd- and analyzed the phylogenetic relationships among pedv strains at the genomic and individual gene levels. the most variable regions were located in the s and orf genes. the s protein is involved in receptor binding and virus entry, the induction of neutralizing antibodies, and host-cell fusion [ ] [ ] [ ] . it is also an important target for monitoring the genetic diversity of coronavirus isolates [ , ] . most pedv reference isolates prior to , including all chinese strains, were partitioned into the first cluster (group ; g ), whereas the three chinese strains (chgd- , bj- - and ch/fjnd- / ) belong to the second cluster (group ; g ). all these three strains were isolated in when severe ped outbreaks were rampant in some areas of china. all korean field strains belong to this cluster (except the japanese isolates kawahira and nk), most of which were isolated in and . interestingly, these three chinese strains formed a unique cluster with the highest amino acid identities to knu- ( . %, . % and . %, respectively). in this study, the same amino acids insertions and deletion of the s gene were observed among three chinese (chgd- , bj- - and ch/fjnd- / ) and two korean (knu- and knu- ) strains. both knu- and knu- were isolated in south korea during - . these results suggest that the recently isolated chinese strains may have originated from korean ones. the orf gene is situated between the s and e genes in the pedv genome, but its function is unknown. previous studies found that a continuous or nucleotide region was deleted within the orf gene when pedv was repeatedly passaged in cell culture, indicating a possible involvement in viral pathogenicity [ , ] . recent investigations demonstrated that all the reported pedv isolates could be classified into three groups, based on phylogenetic analyses of the orf genes [ , ] . our study notably revealed that the amino acid sequence of orf in the chgd- strain was clearly different from those of all other pedv isolates. this difference could not be attributed to the effects of limited cellular passaging, because the orf gene sequence of chgd- passage cells (i.e. intestinal fecal sample) was almost identical to that of passage cells (data not shown). chgd- fell out with the three previously identified groups, and thus constitutes a new group. the chgd- isolate showed . - . %, . %, and . - . % amino acid sequence identities with members of groups , and , respectively. genomic recombination is known to occur at high frequency between heterogenous genomes of coronaviruses. however, recombination analysis provided no evidence to suggest that chgd- was derived directly from the recombination of known pedv strains. further molecular epidemiological evidence is needed to determine the origin and evolution of the chgd- genome. the variations within the chgd- genome, especially in the orf and s genes, place it in a new cluster with bj- - and ch/fjnd- / , based on phylogenetic analysis of the pedv genomes. ped was generally considered to be under control or had only mild effects in swine herds in china before the beginning of . since then, however, ped has unexpectedly devastated many swine farms, including those were killed and attenuated vaccines based on cv were being used, suggesting that these vaccines were no longer able to confer protection. this raised the question of whether any obvious changes in antigenicity and/or virulence are associated with the genetic variations demonstrated in chgd- . the results of this study suggest that there is an urgent need for more fundamental research aimed at understanding the basic biology of this virus strain, as well as the mechanisms of immunogenicity and pathogenesis of pedv. the present study isolated and identified a pedv chgd- strain from infected piglets in guangdong in . analysis of the genetics and evolution of chgd- demonstrated significant genetic diversity compared to other pedv reference strains, suggesting the presence of a new variant pedv in china. outbreaks of diarrhea were observed at a number of pig farms at different locations between january and march . the cases were characterized by watery diarrhea, dehydration and vomiting, with - % morbidity and - % mortality in suckling piglets, whereas affected sows were characterized by diarrhea, anorexia, and depression, but recovered within week. breeding herds had been immunized with attenuated or killed ped-tge combined vaccine produced in china in the fall and winter of the previous year. the pedv component of this vaccine was based on the cv strain. sick or dead piglets aged - days were submitted for laboratory investigation. necropsy examination of all piglets revealed that the small intestines were congested and filled with fluid, and were thin-walled as a result of severe mucosal atrophy. microscopically, marked cytoplasmic vacuolation and exfoliation of enterocytes with subsequent shortening of villi were noted. tissues and feces and/or blood were collected from live or dead animals submitted for polymerase chain reaction (pcr) detection to perform a surveillance. samples from feces and intestine tissues were subjected to virological investigations for common viral swine pathogens such as pedv, transmissible gastroenteritis virus (tgev), porcine rotavirus (prv), porcine circovirus (pcv ) [ ] , porcine kobuvirus [ ] , and porcine bocavirus (pbov) [ ] , using previously described methods. growth medium (gm) was dulbecco's modified eagle's medium (dmem, gibco, usa) supplemented with % heat-inactivated fetal calf serum, . % tryptose phosphate broth (tpb), and antibiotics. maintenance medium (mm) consisted of dmem supplemented with . % tpb and μg/ml trypsin (gibco). virus isolation was performed using vero cells (ccl- ™, atcc), as described previously [ , ] with minor modifications. briefly, intestinal samples positive for pedv by reverse transcription (rt)-pcr were further filtered through a . -μm syringe filter (millipore, usa) and used as inoculum. gm was removed from confluent monolayer cell cultures, which were then washed twice with dmem and inoculated with the filtered intestinal content suspensions. after adsorption for min at °c, the cells were washed with dmem and mm was added. the vero cell cultures were observed for days for cytopathic effects (cpe). immunofluorescence assay (ifa) and electron microscopy were used to detect pedv in the infected cells. the ifa utilized a : dilution of mouse anti-s monoclonal antibody (cat no: , jbt, korea) specific for pedv and a : dilution of fluorescein isothiocyanate-conjugated goat anti-mouse igg (cat no: - - , kpl, usa). for electron microscopy, infected cell culture supernatants were partially purified by ultracentrifugation through a % (wt/wt) sucrose cushion, negatively stained with % ammonium molybdate, and examined with an electron microscope (jem- , jeol ltd., japan). viral rnas were extracted from chgd- -infected vero culture supernatant using trizol ls reagent (invitrogen, usa), according to the manufacturer's instructions. twelve pairs of oligonucleotide primers were used to amplify the different regions of the chgd- genomes, and were designed based on the sequences of pedv strain cv . the pcr products were purified and cloned into pmd -t vector (takara, japan) and sequenced using an automated genome sequence (genetic analyzer xl; applied biosystems, usa). the terminal sequences were acquired using a kit for rapid amplification of cdna ends (race) (clontech, japan). all primers are listed in table . sequence data were assembled and analyzed using lasergene software (dnastar inc., usa). multiple sequence alignments were performed using clustal x . [ ] . phylogenetic analyses were carried out using the mega program [ ] . phylogenetic trees based on the amino acid sequences of the s, orf , m and n proteins were elaborated using the neighbor-joining method, with bootstrapping over , replicates. the pedv strains utilized in the present study including complete genome, s, orf , m and n genes are listed in table , along with their genbank accession numbers. protein kinase-specific phosphorylation sites were identified using the prediction tool kinasephos program through the web server (http://kinasephos.mbc. nctu.edu.tw/predict.php) [ ] . the gene sequence was scanned for possible recombination events using the software package simplot (v . . ), according to the methods described previously [ ] . the genome sequence of chgd- was registered in genbank under the accession number jx . the authors declare that they have no competing interest. authors' contributions yfp, xyt, wl carried out most of the experiments and drafted the manuscript. fc, yhs critically revised the manuscript and the experimental design. qfz, ddw and yzb contributed to the interpretation of the findings and revised the manuscript. all of the authors read and approved the final manuscript. letter to the editor. pig farming a new coronavirus-like particle associated with diarrhea in swine virus-like particles associated with porcine epidemic diarrhoea current aspects in the etiology of viral diarrheas of swine: occurrence of infections with the epizootic viral diarrhea (evd) virus light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (evd): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infections an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan isolation of porcine epidemic diarrhea virus (pedv) in korea molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic 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kobuvirus from pig stool specimens in high prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms in china the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools mega : molecular evolutionary genetics analysis (mega) software version . kinasephos: a web tool for identifying protein kinase-specific phosphorylation sites full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination isolation and characterization of a variant porcine epidemic diarrhea virus in china this work was supported by grants from the special project from guangdong science and technology department (no. b ). submit your next manuscript to biomed central and take full advantage of: key: cord- -a ntxnmm authors: yip, ming shum; leung, nancy hiu lan; cheung, chung yan; li, ping hung; lee, horace hok yeung; daëron, marc; peiris, joseph sriyal malik; bruzzone, roberto; jaume, martial title: antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: a ntxnmm background: public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (sars-cov). we have previously reported that antibodies elicited by a sars-cov vaccine candidate based on recombinant, full-length sars-cov spike-protein trimers, trigger infection of immune cell lines. these observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. methods: we have used primary human immune cells to evaluate their susceptibility to infection by sars-cov in the presence of anti-spike antibodies. fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (rt-pcr) were utilized to assess occurrence and consequences of infection. to gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (fcγr), which bind antibody-coated pathogens. results: we show here that anti-spike immune serum increased infection of human monocyte-derived macrophages by replication-competent sars-cov as well as spike-pseudotyped lentiviral particles (sars-covpp). macrophages infected with sars-cov, however, did not support productive replication of the virus. purified anti-viral iggs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. antibody-mediated infection was dependent on signaling-competent members of the human fcγrii family, which were shown to confer susceptibility to otherwise naïve st cells, as binding of immune complexes to cell surface fcγrii was necessary but not sufficient to trigger antibody-dependent enhancement (ade) of infection. furthermore, only fcγrii with intact cytoplasmic signaling domains were competent to sustain ade of sars-covpp infection, thus providing additional information on the role of downstream signaling by fcγrii. conclusions: these results demonstrate that human macrophages can be infected by sars-cov as a result of igg-mediated ade and indicate that this infection route requires signaling pathways activated downstream of binding to fcγrii receptors. the continuous threat of respiratory viruses to public health was exemplified by the global impact of the sars-cov outbreak in [ ] , by the occurrence since of confirmed human cases of h n avian influenza in many countries, particularly across asia [ ] , and the h n influenza pandemic [ ] . the recent emergence in the arab peninsula of a novel coronavirus responsible for the middle east respiratory syndrome (mers-cov), [ , ] and the new h n strain of avian influenza that has jumped into humans [ , ] in china underscore the need to continue work in this direction. it is now agreed that sars-cov can infect not only the respiratory tract, but can also affect other organ systems and several reports have demonstrated infection of hematopoietic cells [ ] [ ] [ ] ; however, the mechanism by which sars-cov enters into immune cells, which do not express the sars-cov receptor angiotensin-converting enzyme (ace ) [ , ] has remained poorly understood. both c-type lectin receptors such as liver/lymph node-specific intercellular adhesion molecule- -grabbing integrin (l-sign) or dendritic cell specific intercellular adhesion molecule -grabbing non-integrin (dc-sign) [ , ] , as well as antibody-mediated infection may provide sars-cov with an opportunity to modify its tropism. because of the lack of effective antiviral strategies to control coronaviruses infections, vaccination is still regarded as a major option for preventing resurgence of sars and related diseases. we previously showed that a sars-cov vaccine candidate based on recombinant, full-length sars-cov spike-protein trimers triggered infection of human b cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents [ ] . more recently, we demonstrated that anti-spike antibody potentiates infection of both monocytic and lymphoid immune cell lines, not only by sars-covpp but also by replication-competent sars-coronavirus [ ] , thus providing evidence for a novel and versatile mechanism by which sars-cov can enter into target cells that do not express the conventional ace virus receptor and are otherwise refractory to the virus. such infection pathway may have implications for understanding the tropism and pathogenesis of the virus and, therefore, we further investigated the molecular and cellular mechanisms underlying ade of sars-cov infection. by monitoring the susceptibility of human bulk primary immune cells (i.e. peripheral blood mononuclear cells) we have established the occurrence of ade of sars-covpp infection in different circulating immune cell types, among which the monocytic lineage (cd + cells) was the primary target. in addition to monocytes, human macrophages were also infected by sars-cov in presence of anti-spike antibodies only. ade-mediated infection of macrophages, however, did not support productive replication of the virus. finally, we have provided evidence that the intracellular signaling motifbut not the igg binding motifof the fcγr is the key molecular determinant for triggering ade of sars-covpp. our findings conclusively demonstrate that anti-spike serum promotes internalization of sars-cov by human macrophages. primary human macrophages are susceptible to sars-cov infection through antibody-mediated pathway because our previous observations revealed that a human monocytic cell line thp- was susceptible to ade of infection [ ] , we investigated the occurrence of ade of infection in primary human macrophages in vitro, firstly by taking advantage of sars-covpp that can be safely used to mimic the viral entry process [ , ] . we found that sars-covpp opsonized with a : dilution of anti-spike serum readily infected over % of primary human macrophages, as determined by immunofluorescence staining of firefly luciferase at hours post infection (h.p.i.). by contrast, cells exposed to sars-covpp opsonized with control serum did not show any positive staining of the luciferase reporter protein (figure ). these experiments extend our previous observations and indicate that anti-spike antibodies facilitate infection of sars-covpp into human macrophages. we next tested whether this altered tropism was also displayed by replicationcompetent sars-cov. human macrophages were infected at a multiplicity of infection (moi) of with the same dilutions of anti-spike or control immune serum and the infection pattern was examined by both immunofluorescence staining of sars-cov nucleocapsid protein and real-time quantitative pcr measurement of viral rnas. positive immunofluorescence signals were detected only at h.p.i. when sars-cov was opsonized with both control and anti-spike serum ( figure ). interestingly, whereas presence of control serum led to only modest infection by sars-cov (~ % of cells), a -fold increase in the percentage of positive cells was noted in the presence of anti-spike serum for two out of three donors ( figure ). such enhanced infection pattern was paralleled by the number of viral gene copies measured ( figure ). thus, compared to inoculums containing control serum, there was a to -fold increase in the detection of both positive and negative strands of viral genes in macrophages infected in presence of anti-spike serum, which was more pronounced at and h.p.i. there was a rapid decline in viral rna from to h.p.i. for both conditions and no further changes were detected at later time points ( figure ). however, it should be noted that the primer set used for the nucleocapsid gene could also detect the negative strand of the viral genomic rna and could not distinguish it from sub-genomic material. in addition, we measured by real-time quantitative pcr copies of both viral nucleocapsid and orf b genes in culture supernatants to determine the release of sars-cov particles from the infected macrophages. copy numbers of both viral rnas, however, remained unchanged at all the selected time points during the course of the experiment (less than copies/μl), regardless of whether macrophages had been infected in the presence of either control or anti-spike serum (data not shown). we have previously demonstrated that mouse anti-spike serum could trigger infection of raji cells [ ] . in the context of ade, antibodies against viral proteins are considered as the major players of enhancement [ , ] ; see for review [ , ] . to eliminate the possibility of the participation of other soluble factors during ade of sars, in this section we investigated the capability of anti-spike antibodies alone in enhancing infection of immune cells. we purified igg by protein g-sepharose chromatography from mouse anti-spike and control serum, and used fold serial dilutions from to . μg/ml of the purified portion to form immune complex with sars-covpp and then infected raji cells. our results show that purified igg from mouse anti-spike serum triggered infection in raji cells, which was more pronounced with increasing immunoglobulin concentrations ( figure ). the flow-through (ft) from the same serum, which was diluted by the appropriate factor to be comparable to the final igg concentration used, elicited no detectable infection at all concentrations (data not shown). significant differences between ade of infection with purified mouse anti-spike igg and flow-through were observed at concentrations and . μg/ml, and marginal difference was observed at . μg/ml. as expected, neither purified igg ( figure ) nor flow-through from mouse control serum triggered significant ade of infection at all concentrations. our recent work had revealed the predominant role of human fcγrii (cd ) in mediating ade of sars-cov [ ] . to gain further insight into the underlying molecular mechanism we have investigated the involvement of different domains of fcγrii in mediating ade. to this end we produced a series of truncated constructs that only carried ectodomain and transmembrane domains of fcγrii, and chimeric constructs with the ectodomain of one receptor and transmembrane and endodomain of another ( figure a ). we then verified the expression of these constructs on st cells by flow cytometry using a specific monoclonal antibody (clone fli . ) that binds to the second ig-like domain d γ of fcγrii. of note, the fc binding portion of the fcγrii group is very similar among fcγriia-h , fcγriia-r , fcγriib [ ] and also fcγriib , as it harbors identical extracellular structures as fcγriib . our findings indicate that all fcγrii constructs exhibited detectable expression of fcγrii (dark grey) in st cells in comparison to isotype control (light grey) ( figure b ). we then tested the ability of the various constructs to bind purified anti-spike igg-sars-covpp immune complexes and observed that all st transfectants were able to bind immune complexes ( figure c ). finally we investigated whether any difference in susceptibility to sars-covpp ade of infection was conferred by different fcγrii constructs. when immune complexes formed by μg/ml of purified mouse anti-spike igg (the concentration at which the highest infection level was observed in raji cells) and sars-covpp were added to fcγrii-expressing st cells, all four transfectants expressing wildtype fcγrii forms (cf. fcγriia-h vs. fcγriia-r, and fcγriib vs. fcγriib , corresponding to constructs , , , , respectively) were infected ( figure d ), with fcγriia-expressing st being more prone to infection than fcγriib (cf, constructs and with ). all the endodomain-truncated constructs (fcγriia-h.Δic, fcγriia-r.Δic and fcγriib.Δic, corresponding to constructs , , respectively) were not susceptible to ade of infection, indicating that binding of anti-spike igg-sars-covpp immune complexes was not sufficient to mediate entry and that the signaling-competent endodomain was required. however, not all chimeric constructs were able to sustain ade of infection, suggesting that domain swapping may have partially interfered with signal transduction. thus only chimeras with fcγriia-h ectodomain and fcγriib endomain, or with fcγriib ectodomain and fcγriia endomain, exhibited a statistically significant ade of infection ( figure d ). this cannot be explained by differences in surface expression or binding of opsonized pseudoparticles, as fcγriib.ec/ iia.ic (viz., construct ) showed robust ade despite one of the lowest binding ability of immune-complexes. the possibility that immune response to pathogens may have also deleterious effects on the host homeostasis has been the focus of several studies. for example, the hyper-induction of cytokines following avian influenza infection has been implicated in the severity of the disease [ ] and infection of cells by ade has been known to occur for several viral diseases [ , ] . here we demonstrate that anti-spike antibody potentiates infection although we unambiguously obtained evidence of ongoing infection (e.g., de novo synthesis of the structural viral protein n), ade-infected macrophages did not support productive replication of sars-cov and, after initiation of viral gene transcription and viral protein synthesis, the replication process stalled ultimately ending in an abortive viral cycle without detectable release of progeny virus. abortive replication of sars-cov into macrophages has already been documented [ ] but, at variance with this previous report in which % of the macrophages were infected by sars-cov in the absence of immune-serum (moi = - ), we observed a much lower infection rate (about - %). one possibility is that such discrepancy may be due to difference of time of sampling ( hours in our study versus hours in cheung's study) and the protocol used for in vitro differentiation (viz., macrophages were differentiated in the presence of fetal calf serum in our study, and autologous plasma was removed two days prior to infection in ref. [ ] ), leading to difference in infectivity of the cells observed in the studies. alternatively, we should also consider that the readout of the pseudo-particle experiments was the expression of the luciferase reporter gene, which is under the control of the hiv backbone. this may lead to higher level of protein expression when compared to the abortive replication that follows sars-cov infection of macrophages. thus, the difference may be, in part due to the inability to detect low amounts of spike protein by immunofluorescence and the difference in sensitivity of the two methods. of note, the anti-spike mediated entry is specific for spike-pseudotyped particles, as shown in figure of [ ] . because clinical observations have reported poor disease outcomes in early seroconverted sars patients [ ] [ ] [ ] , it would be of interest to test sars patient sera collected at different time-points after sars onset. however, we have been unable thus far to conduct conclusive assays on a well characterized serum library; moreover, we have to be cognizant of the possibility that ade may only occur within a narrow window during the course of an infection and only in a subset of infected patients. the alternative possibility that internalization by macrophages may in fact represent an additional mechanism to control viral spread requires further investigation. in general, studies aiming at better understanding antibody-dependent enhancement of viral infections are focusing on either identifying the (immune) receptor(s) and/or serum component(s) allowing penetration of the pathogen into the target cell, also known as extrinsic ade, or the outcome response(s) of the target cell downstream to ade of infectionor intrinsic ade [ , , ] . our previous results demonstrated that only fcγriia and, to a lesser extent, fcγriib triggered infection by sars-covpp in presence of anti-spike serum [ ] . although it would have been desirable to perform fcγr blocking experiments also in macrophages, coexpression of all fcγrs in these cells [ , ] however, the relationship between internalization of immune complexes and ade of infection by sars-cov via fcγriis appears to be a complex process. thus, fcγriib has been shown to mediate internalization of immune complexes at a faster rate than fcγriia [ ] , whereas we found that ade of infection via fcγriia was more prominent than with fcγriib. recently, involvement of downstream signaling triggered by fcγrs activation has been evaluated with respect to ade of dengue virus infection [ ] . thus, abrogation of fcγri and fcγrii signaling competency was associated with significant impairment of phagocytosis, but only the signaling-incompetent fcγri become unable to trigger ade of dengue virus. conversely, no discernible effect on dengue virus immune complex infectivity was observed for both wild type and signaling incompetent fcγriia. these findings point to fundamental differences between fcγria and fcγriia with respect to their immuneenhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these fcγrs [ ] . altogether our results demonstrate that, in presence of vaccine-elicited antiviral antibodies, sars-cov displays an altered tropism toward primary human immune cells, which do not express the conventional virus receptor and are otherwise refractory to the virus. a number of sars vaccine candidates have been tested in experimental animal models [ , ] , many of them based on the viral spike glycoprotein previously identified as the most immunogenic antigen inducing neutralizing and protective antibodies [ , , , ] . of note, some vaccines against animal coronaviruses have been also generated, but their development has proven difficult due to immune [ ] [ ] [ ] . despite the fact that this alternative infection pathway appears to have a limited impact, it remains of interest to appreciate the cost of antibody-mediated sars-cov infection on the functionality of the target cells, in order to have a broad understanding of the tropism and pathogenesis of the virus and evaluate potential pitfalls associated with immunization against human coronaviruses. this aspect is gaining more relevance as the emergence of the mers-cov further indicates that the availability of vaccines targeting this group of viruses, which have demonstrated the ability to jump species, is one of the few options available to prevent the spread of infections causing severe diseases with high mortality in humans [ ] . the following cell lines used in the study were obtained from the american type culture collection (atcc; manassas, va, usa): hek t (human kidney epithelial cells; crl- ), raji (burkitt's lymphoma/b lymphoblast), st (burkitt's lymphoma/b lymphoblast lacking expression of fcγr; crl- ). hek t cells were cultured in dmem supplemented with % heat-inactivated fetal bovine serum (fbs), . mg/l penicillin and mg/l (hfcγriib ) . b) surface expression in stably transduced st cell lines was evaluated by flow cytometry using a mouse monoclonal anti-fcγrii antibodies (dark grey) or isotype control (light grey), and expressed as mean fluorescence intensity in arbitrary units (au) as described under materials and methods. constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. c) immune complex-binding ability of wild-type, truncated and chimeric fcγr receptors. sars-covpp were incubated with purified mouse anti-spike igg (dark grey) or control igg (light grey) to form immune complexes, which were then added to the st transfectants (see materials and methods). after washing and fixation, bound immune complexes were detected by flow cytometry with an fitc-conjugated goat anti-mouse f(ab') , and results shown as in b. constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. d) susceptibility of st transfectants to ade of infection by sars-covpp. sars-covpp were incubated with purified mouse anti-spike igg (dark grey) or control igg (light grey) and then used to infect cells (see materials and methods). cells were washed and three days post infection incubation was stopped by adding luciferase substrate to measure enzymatic activity, which was expressed as relative luminescence units (rlu). constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. results are shown as the means ± sem of three (b and c) or - independent experiments (d). *p < . ; **p < . by the unpaired student's t test. streptomycin, whereas hematopoietic cells were grown in rpmi- supplemented with % heat-inactivated fbs, % non-essential amino-acids, mm l-glutamine, mm sodium pyruvate, . mg/l penicillin, mg/l streptomycin, and μm -ß-mercaptoethanol (all from invitrogen life technologies, carlsbad, ca, usa). all cells were maintained at °c in a humidified atmosphere with % co supply. the research protocol was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw - ). blood samples of healthy donors were obtained from the hong kong red cross blood transfusion service. human blood cells were isolated from buffy coats and monocyte-derived macrophages were generated in vitro using a modified protocol as previously described [ , ] . briefly, mononuclear cells were isolated by a ficoll-paque density gradient (pharmacia biotech, uppsala, sweden) to remove erythrocytes, granulocytes, and cell debris. monocytes were enriched by plastic adherence, harvested, seeded ( cells/ml) on tissue culture plates and allowed to differentiate for days in the presence of % autologous human plasma and % fetal calf serum before use. the purity of macrophages was consistently above %, as ascertained by immunofluorescence staining for human cd , a lysosomal glycoprotein that is highly expressed by macrophages and monocytes [ , ] . balb/c mice were immunized with recombinant sars-cov spike proteins adjuvanted with alum, as previously described [ , ] . saline-injected mice served as controls. serum was collected at day post-immunization, heatinactivated for min at °c and stored at − °c for subsequent use. the protocol to produce sars-cov pseudotyped lentiviral particles expressing a luciferase reporter gene (sars-covpp) has been described elsewhere [ ] . following a purification step on % sucrose cushion, the concentrated viral particles were titrated by elisa for lentivirus-associated hiv- p protein according to the manufacturer's instruction (cell biolabs, inc., san diego, ca, usa), and stored at − °c until use. for ade assays, heat-inactivated mouse anti-spike or control serum was incubated for hr at °c with sars-covpp. this inoculum was deposited on cells and infection proceeded for hr at °c. following repeated washing cells were incubated for additional - hours and then fixed in % paraformaldehyde (sigma-aldrich inc., st. louis, mo, usa) for immunofluorescence microscopy. laboratory procedures involving replication-competent viruses were performed in biosafety level- containment (state key laboratory of emerging infectious diseases, the university of hong kong). the sars-cov strain used (hk ) is a clinical isolate [ ] , which was cultured in fetal rhesus kidney- (frhk- , atcc code crl- ) cells. in ade assays, heat-inactivated mouse anti-spike or control serum was incubated for h at °c with sars-cov and human monocyte-derived macrophages were infected at an moi of for min at °c. after repeated washings, cells were cultured with fresh medium (time ) supplemented with % fbs for the indicated time points (hours) post infection (p.i.) and eventually either fixed in % paraformaldehyde (sigma-aldrich inc.) for immunofluorescence microscopy, or resuspended in lysis buffer (rlt buffer, rneasy rna mini kit; qia-gen, germantown, md, usa) for real-time quantitative rt-pcr. samples of cell culture supernatants harvested at different h.p.i. were also mixed with rlt buffer and processed as above for rt-pcr. infectivity of replication-competent sars-cov was determined by indirect immunofluorescence with a mouse monoclonal antibody directed against sars-cov nucleocapsid protein (clone d ; a gift from dr. kwok-hung chan, department of clinical microbiology, queen mary hospital, hong kong), at a dilution of : . staining was revealed with a tetramethylrhodamine- -(and- )isothiocyanate (tritc) conjugated goat anti-mouse antibody (invitrogen life technologies) on an axioobserver z microscope (carl zeiss, inc., thornwood, new york, usa). pictures taken from randomly chosen fields at a magnification of x were acquired with an axiocam mrm camera and analyzed with metamorph software (molecular devices, sunnyvale, ca, usa). infectivity was determined as the percentage of cells expressing viral antigen. similar staining procedures were employed for assessing infection by sars-covpp, except for the use of direct immunofluorescence with a fluorescein isothiocyanate (fitc) conjugated goat monoclonal antibody specific for firefly luciferase (rockland, gilbertsville, pa, usa) at a dilution of : . rna was extracted with the rneasy rna mini kit (qiagen), according to the manufacturer's recommendations; concentration and purity of rna were measured by standard optical methods. reverse transcription was performed on total rnas with superscript iii reverse transcriptase as specified by the manufacturer (invitrogen life technologies). for quantification of sars-cov gene copies, cdna was generated in separate reactions in presence of either forward or reverse primers specific for the nucleocapsid and orf b genes ( table ) that amplified negative and positive strand viral rnas, respectively. this assay allows distinguishing between signals generated by entry of input virus from downstream events reflecting an active replication process [ ] . specific primers (table ) and taqman minor groove binder probes used for detection of sars-cov nucleocapsid gene have been previously described [ ] . however, it should be noted that the primer set used for the nucleocapsid gene could also detect the negative strand of the viral genomic rna and could not distinguish it from sub-genomic material. the qpcr assay was carried out in a final volume of μl and the fluorescence signal was detected with a lightcycler -ii (roche applied science, mannheim, germany) programmed as follows: °c for min, followed by cycles of °c for sec, °c for sec, and °c for sec. results were expressed as the number of target copies per copies of the s rrna gene, which was used to normalize results. to ensure the consistency of qpcr measurements over the time period of the study, the same batches of primers and probes were employed. the qpcr results were considered valid when the efficiency of the standard curve was between . to . and the r value was greater than . . iggs was purified from immunized and control mice using protein g-sepharose (ge healthcare, little chalfont, united kingdom) according to the manufacturer's recommendations. the purity of the igg fraction was checked by silver staining following gel electrophoresis and the concentration of anti-spike igg determined by elisa. construction of lentiviral vectors for wildtype, endodomain-truncated and chimeric forms of fcγrii (hcd ) the strategy to produce plasmids for wild-type human igg receptors (fcγrs) consisted in replacing the enhanced green fluorescent protein (egfp) gene from the bicistronic vector pchmws-egfp_ires_hygromycin (a kind gift from drs. rik gijsbers and zeger debyser, katholieke universiteit leuven, belgium) with the coding sequences for fcγriia (hcd a) isoforms (fcγriia.r and fgriia. h genbank accession no. nm_ ) and fcγriib (hcd b; genbank accession no. af ) flanked by bglii and sali sites, as described elsewhere [ ] . the synthetic sequences (geneart, regensburg, germany) were inserted into the original transfer plasmid to yield wildtype constructs. pchmws-fcγriib _ires_hygromycin was constructed by swapping the intracellular domain of fcγriib , obtained from rt-pcr amplification of the desired sequence from polyclonal raji cdna, with the corresponding region of the pchmws-fcγriib _ires_hygromycin construct. wild-type constructs were subsequently used to generate truncated and chimeric forms of fcγriis by standard techniques (see figure a for a schematic representation of the mutants tested). all plasmids were sequenced by the centre for genomic sciences of the university of hong kong. generation of stable st cell lines expressing wild-type and mutated fcγrs using lentiviral particle-based gene transduction cell lines were generated by transduction of monoclonal st cells with vesicular stomatitis virus (vsv) g pseudotyped lentiviral particles bearing the specified transgene as previously described [ ] . at days post-infection, cell surface expression of the fcγrs was monitored by flow cytometry and cells were subsequently cultured in selective medium containing μg/ml hygromycin (invitrogen life technologies) when appropriate. finally, several monoclonal cell lines for each construct were isolated and expression of the transgene was confirmed by flow cytometry. expression of fcγriis was evaluated by flow cytometry as described in [ ] . the following mouse mabs were table sequences of primers and probes used in real-time qpcr assay and cdna synthesis used: g anti-hcd , fli . for hcd , and . anti-hcd (all from bd pharmingen, frankin lakes, nj, usa); mopc- (igg , κ) or mpc- (igg b, κ) isotype controls (both from biolegend, san diego, ca, usa). cells were washed and primary antibody binding was revealed by staining on ice for min with fitcconjugated goat anti-mouse antibodies (jackson immu-noresearch, west grove, pa, usa). data were collected from at least , singlet living cells on a lsrii flow cytometer (bd biosciences, san jose, ca, usa) and analyzed using flowjo software (treestar, ashland, or, usa). sars-covpp-igg immune complexes were obtained by incubating sars-covpp with μg/ml of either purified mouse anti-spike or control igg at °c for hr. the mixture was then quickly chilled on ice for min and added ( μl/well) to st cells ( × cells/well), which had been previously stained with . % fixable viability dye efluor (ebioscience, san diego, ca, usa). following a hr incubation on ice, cells were washed twice with cold pbs, fixed with % paraformaldehyde for min on ice and immune complex binding was revealed by staining with μg/ml fitc-conjugated goat anti-mouse f(ab') (jackson immunoresearch) at °c for min. data were collected and analysed as described above. results are shown as means ± sem of the indicated number of observations. statistical difference between groups was determined by the unpaired students's t-test with a . significance level. severe acute respiratory syndrome writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza av an influenza a h n virus revivalpandemic h n / virus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia human infection with a novel avian-origin influenza a (h n ) virus origin and diversity of novel avian influenza a h n viruses causing human infection: phylogenetic, structural, and coalescent analyses multiple organ infection and the pathogenesis of sars sarscoronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients sars-coronavirus replication in human peripheral monocytes/macrophages tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway cleavage of the sars coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages mechanisms and results of the antibody-dependent enhancement of viral infections and role in the pathogenesis of coxsackievirus b-induced diseases antibody-mediated enhancement of viral disease antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications mapping epitopes of human fc gamma rii (cdw ) with monoclonal antibodies and recombinant receptors the role of influenza virus gene constellation and viral morphology on cytokine induction, pathogenesis, and viral virulence cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis neutralizing antibody response and sars severity anti-sars-cov igg response in relation to disease severity of severe acute respiratory syndrome antibody responses against sars coronavirus are correlated with disease outcome of infected individuals intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes fc receptor biology fcgamma receptors as regulators of immune responses specificity and affinity of human fcgamma receptors and their polymorphic variants for human igg subclasses biology of immunoreceptor tyrosine-based inhibition motif-bearing molecules differences in endocytosis mediated by fcgammariia and fcgammariib differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human fcgamma ria (cd ) or fcgammariia (cd ) the spike protein of sars-cov-a target for vaccine and therapeutic development vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity osterhaus ad: vaccine-induced enhancement of viral infections animal coronavirus vaccines: lessons for sars immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens the middle east respiratory syndrome-how worried should we be? mbio induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars molecular cloning of cd , a human macrophage marker related to lysosomal glycoproteins clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus the hku-pasteur research pole is a member of the institut pasteur international network. we wish to thank lewis siu (hku-pasteur research pole) for sharing with us his stock of sars-cov spike lentiviral particles when it was most needed. we are grateful to kwok hung chan (queen mary hospital, pokfulam, hong kong) for the gift of the mouse monoclonal antibody ( d ) specific for the sars-cov nucleoprotein, and rik gijsbers and zeger debyser (katholieke universiteit leuven, belgium) for providing the bicistronic retroviral vector used to establish the fcγr-expressing stable cell lines. many thanks to suki lee, chris mok and members of the hku-pasteur research pole for their expert advice and critical reading of the manuscript. m.s. yip was a phd and nancy h.l. leung was an mphil student supported in part by the university of hong kong. this work was supported by the research fund for the control of infectious disease (rfcid; project no. ) of the hong kong government and by the respari project of the institut pasteur international network. the authors declare that they have no competing interests. key: cord- -be qely authors: lyoo, hey rhyoung; park, soo young; kim, ji young; jeong, yong seok title: constant up-regulation of bip/grp expression prevents virus-induced apoptosis in bhk- cells with japanese encephalitis virus persistent infection date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: be qely background: persistent infection of the japanese encephalitis virus (jev) has been reported in clinical cases, experimental animals, and various cell culture systems. we previously reported the establishment of spontaneous jev persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in bhk- cells devoid of virus-induced apoptosis, cbs - and cbs - . however, cell-specific factors may play important roles in controlling jev replication and have never been assessed for this specific phenomenon. recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (upr). results: to identify the host cell factors that affect jev persistence, we investigated the expression of essential upr factors in cbs - and cbs - cells. of the selected upr factors tested, the most noticeable deviations from those of the normal bhk- cells with jev acute infection were as follows: the suppression of c/ebp homologous binding protein (chop) and the constant up-regulation of immunoglobulin binding protein (bip) expression in cbs - and cbs - cells. in jev acute infection on normal bhk- cells, silencing chop expression through specific sirna blocked cell death almost completely. meanwhile, depletion of bip by specific sirna unlocked chop expression in cbs - and cbs - cells, resulting in massive cell death. fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the jev persistently infected cells still contained functional arms for cell fate decisions. conclusions: bhk- cells with jev persistent infection strive against virus-induced apoptosis through constant up-regulation of bip expression, resulting in the complete depletion of chop even with apparent virus amplification in the cells. accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of jev persistent infection in mammalian cells. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. viruses have evolved a wide range of strategies to persist in their hosts. it remains a challenge to understand the mechanisms whereby viral persistence is established and maintained, especially viral persistence within a cell or group of cells. mechanisms by which rna virus persistence is initiated and maintained usually involve two virus-specific factors: the generation of defective interfering (di) particles or temperature-sensitive mutation of wild-type virus [ , ] . research suggests that host factors involved in the control of persistent infection relate to elements of innate immunity in morbillivirus [ ] and cellular protein synthesis in reovirus [ ] . protein synthesis and folding occurs in the endoplasmic reticulum (er). mammalian cells have evolved many sophisticated signaling pathways to monitor any abnormality, including the accumulation of misfolded proteins; these pathways are known as the unfolded protein response (upr) [ ] . these signaling pathways monitor the er's capacity to refold and/or remove abnormally folded proteins and to make cell-fate decisions according to the homeostatic balance [ , ] . in all known animal cells, the following are known to be activated to initiate the upr: three er-localized transmembrane upr transducers, inositol requiring kinase (ire ), double-stranded rnaactivated protein kinase-like kinase (perk), and activating transcription factor (atf ) [ ] . under basal conditions, these three sensors are associated with immunoglobulin binding protein (bip), also known as grp , which is a chaperone of the heat shock protein family. each branch operates parallel with a particular target downstream and contributes to both cell-protective and celldeath pathways [ , ] . under severe or chronic er stress, the upr switches its mode of action toward apoptosis. c/ebp homologous binding protein (chop), also known as growth arrest and dna damage-inducible protein (gadd ), is the pro-apoptotic transcription factor that plays an important role in regulating cell death after er stress [ , ] . several molecular mechanisms of chopinduced apoptosis have been cited, such as compromised alteration of bcl- family proteins [ , ] . a variety of viruses induce er stress and the upr, having evolved various mechanisms to cope with the upr [ ] . west nile virus modulates all three arms of the upr and induces numerous apoptotic responses, including induction of chop expression [ ] . modulation of the upr by the west nile virus is regulated differentially along with its replication cycle [ ] . similar to other flaviviruses, the dengue virus also induces the three arms of the upr and chop expression. however, activated chop does not induce its downstream apoptotic markers, such as suppression of anti-apoptotic protein bcl- and activation of caspase- or caspase- [ , ] . in addition, studies of the hepatitis c virus have shown that both viral structural (envelope) and non-structural (ns ) proteins can induce er stress and the upr activation with up-regulation of bip and chop [ , ] . japanese encephalitis virus (jev), a member of the flaviviridae, is the causative agent of encephalitis in humans and can be transmitted by persistently jevinfected mosquitoes [ ] . viral persistence in the nervous systems of jev-infected patients has been shown in approximately % of jev cases, suggesting that jev persistence may contribute to neural sequelae after acute infection [ ] . though jev is usually cytolytic for susceptible cells, persistent infection of jev has been established in various cell cultures, including baby hamster kidney (bhk)- [ ] [ ] [ ] [ ] [ ] [ ] [ ] as well as in a mouse model [ ] . the underlying mechanisms for jev persistence in cultured cells are not clearly described. we have previously demonstrated spontaneous establishment of persistent jev infection in bhk- cells via serial undiluted passages without any supplemental treatment [ ] . examples of supplemental treatment include bcl- overexpression [ ] or indirect infection with supernatants from persistently jev-infected mosquito cells [ ] . our previous study suggested that di particle accumulation and helper virus attenuation are possible mechanisms for the development and maintenance of jev persistence in bhk- cells. nonetheless, labile cellular factors that may play a role in jev persistence have never been identified in this system. jev also induces er stress and the upr, and studies suggest that the activation of the upr is a major cause of jev-induced apoptosis [ ] . the upr has never been assessed in jev persistent infection, however. in this study, we utilized two persistently jevinfected bhk- cell clones (previously reported in [ ] ) to identify the cell-specific factors of the upr involved in jev persistence in mammalian cells. these cell clones seldom, if ever, undergo apoptosis, while jev replicates actively within. we observed that there was no chop expression at any time, but a significant amount of bip expression was constant in these cells. knockdown of bip expression resulted in chop induction and subsequent cell death. because the level of jev amplification in these cells was not low enough to hold an apoptotic process, we suggest that the readjustment of bip expression in host cells could be one of key factors involved in cell fate decision under viral persistence. many viruses that are originally cytopathic have been found to lose their cytopathicity when the persistent infection is established [ ] . jev infection induces severe cytopathic effects in various cell culture systems, including bhk- cells, and researchers have documented the er stress response and subsequent apoptosis in the jevinfected cells [ , ] . in order to assess how much of the jev persistently infected bhk- cell population is destined to apoptosis while continuously producing infectious virus particles, the two bhk- cell clones with jev persistent infection-cbs - and cbs - -were subjected to flow cytometry analysis after annexin v/propidium iodide staining. the number of apoptotic cells and the late apoptotic or necrotic cells increased significantly upon wild-type jev infection in normal bhk- cells ( figure a ). on the contrary, the number of cbs - or cbs - cells with jev persistent infection undergoing apoptosis seemed to remain below the basal level shown in the naïve bhk- cells. the amount of intracellular infectious jev particles produced in the persistently infected cells was -to -fold less than that of an acute infection ( figure b ). all cells subjected to the flow cytometry were plated and analyzed at the same time points. the cells were grown under the same culture conditions, and no notable difference in the cell numbers of each population was observed. constant expression of elevated amounts of bip and complete depletion of chop were associated with the survival of the persistently infected bhk- cells jev infection is known to induce the upr in bhk- cells through bip-perk and/or bip-ire arms, which is followed by chop-mediated apoptosis [ , , ] . based on these reports, this study primarily assessed the expression profile of the upr factors in the bip-perk arm of the bhk- cells with jev persistent infection. normal bhk- cells were infected with jev and harvested at , , , , , , and hr post-infection (p.i.) in order to obtain cell lysates. two jev persistently infected bhk- cell clones, cbs - and cbs - , were freshly seeded and harvested at their confluence on the culture flask; their cell lysates were subjected to western blotting. phosphorylation of perk was gradually increased, peaked at hr p.i., and decreased thereafter in acutely jev-infected cells ( figure a ). in persistently jev-infected cells, however, there was only a minute amount of both perk and p-perk. unlike perk, the expression of eif α in the persistently infected cells seemed enhanced, as shown in normal bhk- cells with jev acute infection at - hr p.i.; however, the amount of p-eif α was barely detectable (figure a) . phosphorylation of eif α often leads to inhibition of protein translation in general, but the translation of atf is promoted by p-eif α [ ] . this phenomenon was reconfirmed in this experiment as the expression of atf increased gradually hr p.i. in jev acutely infected cells (figure a) . chop, the key mediator of er stressinduced apoptosis, was also induced and accumulated gradually along with the atf activation. in contrast, for the jev persistently infected cell clones, the expression of chop was not detected at all, even in the presence of atf for its transcription (figure a ). unlike cbs - , impaired expression of atf in cbs - was repeatedly noticed in several independent experiments. it was noteworthy that a much higher level of bip was maintained in both jev persistently infected cells throughout the culture period. to further investigate whether the jev persistently infected cell clones kept the upr pathway intact, naïve bhk- cells and the two cell clones with jev persistent infection were treated with . μg tunicamycin ml − for hr. compared to the dmso-treated control, both tunicamycin-treated normal bhk- cells and cbs - cells showed perk-eif α-atf pathway activation followed by chop induction ( figure b ). most of the cells treated with tunicamycin, including cbs - and cbs - cells, succumbed to apoptotic cell death within hr (data not shown). although chop was also clearly induced, expression of p-perk or atf in cbs - cells was not comparable to the normal bhk- cells or cbs - cells on tunicamycin treatment for unknown reasons. this observation suggests that cbs - cell clones may utilize another pathway to induce chop expression, perhaps involving ire activation. these differences in the activation process of the bip-perk arm between cbs - and cbs - imply that individual cells comprising a cell batch with jev persistent infection could have their own unique modification in cellular physiology to avoid the virus-induced apoptosis. taken together, these results suggest that the jev persistently infected cells avoid fulminant apoptosis by maintaining a constant, highly-elevated level of bip, which results in the complete suppression of chop induction. based on the observations, there was no detectable chop expression during continued virus replication in the jev persistently infected cells ( figures b, a , and additional file : figure s ). therefore, we attempted to clarify the effects of chop on jev-induced apoptosis and on virus replication efficiency. naïve bhk- cells transfected with specific sirna for chop were infected with jev at hr post-transfection and harvested at hr p.i. the cell lysates were examined by western blotting with antibodies against chop, bcl- , caspase- , and jev ns . the expression of chop was efficiently silenced, and cleavage of caspase- was not detected in sichop-transfected cells ( figure a ). we also found that, even in the reduced expression of bcl- , sichoptransfected cells were highly resistant to the jev-induced cytopathic effect, as measured by trypan blue exclusion ( figure a and b) . the level of jev ns protein in the cells transfected with sichop and then infected with jev was slightly lower than that of the other cells transfected with scramble sirna ( figure a ). this observation aligns with the result that the virus titer obtained from the chopsilenced cells was slightly compromised compared to those from the control cells at hr p.i. (figure c ). these results are also consistent with a report noting that up-regulation of chop during jev infection plays a key role in virus-induced apoptosis [ ] . as infectious bronchitis virus-induced apoptosis was suppressed and virus replication inhibited in the chop-knockdown cells [ ] , the complete blockage of chop expression in cbs - or cbs - cells might intervene in jev replication and therefore assist in the development of jev persistent infection. however, a clear explanation for this observation is presently beyond the scope of this study. in this study of jev persistently infected cells compared to an acute infection in normal bhk- cells, the most distinguishable aspects regarding the upr factors were the complete depletion of chop and the constant upregulation of bip expression (figure a and additional file : figure s ). some previous studies also showed that bip overexpression attenuates er stress signaling and is protective against apoptosis [ ] . therefore, we decided to assess the implications of the constant expression of bip for maintaining cell viability against virus-induced apoptosis in the jev persistently infected cells. the cbs - and cbs - cells were transfected with specific sirna for bip and harvested hr later. the effect of bip silencing was examined by western blotting with antibodies against bip, chop, caspase- , and jev ns . the expression of bip was suppressed almost completely by sibip in both cbs - and cbs - cells, while the chop expression was clearly induced ( figure a ). in addition, the numbers of viable cells decreased significantly in both cell lines according to cleavage of caspase- ( figure a and b) . the results suggest that the constant overexpression of bip in the jev persistently infected cells somehow holds back chop expression, resulting in the prevention of virus-induced apoptosis. this observation is consistent with reports that the inhibition of chop guarantees a higher survival rate both in vivo and in vitro even though chop is not the sole factor promoting cell death undergoing er stress [ , ] . furthermore, these results revealed that resistance against virus-induced apoptosis of the cbs - and cbs - cells did not result from the lower level of virus replication efficiency; rather it was ascribable primarily to the active participation of cellular factors in the upr. in conclusion, bhk- cells with jev persistent infection strive against virus-induced apoptosis through constant up-regulation of bip expression, a key chaperone involved in er stress. as demonstrated in tunicamycin treatment, these cells maintained their capacity to decide their own cell death fate by inducing chop, although some of the upr factors relaying the bip-perk-atf arm appeared to be impaired. in a previous study, we successfully established jev persistent infection in several mammalian cells in the presence of di particle generation [ ] . certain modifications in the genetic makeup of helper jev could also play a role in the development and maintenance of the viral persistent infection [ ] . therefore, the observations made in this experiment demonstrate that both the compromised virus replication capacity and certain cellular factors, such as the upr factors, also participate in establishing viral persistent infection in mammalian cells. our study highlights the importance of certain host cell factors in er stress-signaling pathways for jev persistence by utilizing an in vitro model for the first time. this work provides new insight into the complex mechanism of viral persistence and potentially contributes to developing useful agents and tools for therapeutic intervention in the clinical sequelae of japanese encephalitis. baby hamster kidney- (bhk- ; korea cell line bank) cells were maintained in a minimum essential medium (mem; gibco) containing % fbs (gibco) and units of penicillin-streptomycin (gibco) ml − . the persistently jev-infected bhk- cells have been described previously [ ] . two cell clones, cbs - and cbs - , were chosen for use. all cells were grown at °c in a % co incubator. jev k p strain (provided by the korean national institute of health) was employed throughout this study. propagation of the virus was carried out in bhk- at °c in mem supplemented with % fbs for hr. after infection, the virus-containing supernatant was collected and centrifuged to remove cell debris, and then stored at − °c. the virus was inoculated on a monolayer of bhk- cells in mm plates; an overlay medium was applied containing % fbs, % penicillin-streptomycin ( , u), % × mem, % × mem, and % agarose. after to days incubation, the cells were fixed with . % formaldehyde in phosphate buffered saline (pbs) for hr and stained with . % crystal violet. to titrate the intracellular virus particles, the infected cells were washed with pbs, trypsinized, and resuspended in ml of mem. after three times of freeze and thaw cycles, the cell debris was pelleted before collecting the virus-containing supernatant. the virus titer was determined by a plaque-forming assay. to analyze apoptosis, an annexin v-fluorescein isothiocyanate (fitc) and pi double-staining method (meb-cyto apoptosis kit; mbl) was used according to the manufacturer's protocol. after the adherent cells were harvested, they were re-suspended in binding buffer, and μl annexin v-fitc and . μl pi were added to the cell samples. the mixtures were incubated for min in the dark at room temperature and then analyzed by flow cytometry (bd facscalibur). tunicamycin (sigma-aldrich) was dissolved in dmso. rabbit anti-ns antibody was kindly provided by professor radhakrishnan padmanabhan (georgetown university, usa). antibodies against p-perk, total perk, p-eif α, total eif α, bip, and caspase- were purchased from cell signaling technology. antibodies against chop and bcl- were purchased from santa cruz biotechnology. anti-atf antibody and anti-β-actin antibody were sourced from abcam and neomarkers, respectively. hrpconjugated goat anti-mouse antibody and hrp-conjugated goat anti-rabbit antibody were obtained from molecular probes and invitrogen, respectively. the bhk- cells, cbs - and cbs - , were seeded in -well plates and grown to % confluence. sichop, sibip, and scramble sirna were purchased from santa cruz. transfection of sirna was conducted using jet-prime™ (polyplus-transfection) according to the manufacturer's instructions. the bhk- cells were infected with jev at hr post-transfection. the cells and the supernatant were harvested at hr post-infection, and the pi cell clones were harvested at hr posttransfection for further analysis. the collected supernatant was used for the quantification of the viral production, and viable cells were counted using trypan blue exclusion. for total protein extraction, virus-or mock-infected cells in monolayers were washed with cold pbs and then lysed in ice-cold m-per buffer (pierce) with a cocktail of protease inhibitors (roche). proteins were separated with . % or % gradient page using the gradi-gel™ gradient analysis kit (elpis biotech); they were subsequently transferred to pvdf membrane (millipore). the membrane was blocked with % skim milk in tbst. primary antibodies were incubated at °c with membrane overnight in % skim milk in tbst or % bsa in tbst. after primary incubation, the membrane was washed in tbst once for min and three times for min; it then was incubated with secondary antibodies in % skim milk in tbst at room temperature for hr. the membrane was washed again three times in tbst for min, and the proteins were detected with an enhanced luminol-based chemiluminescent detection kit (abfrontier) according to the manufacturer's instructions. the intensities of bands from western blot analysis were quantified using the imagej program (national institutes of health) according to the developer's instructions. the data were presented as mean ± standard deviations (sd) of three independent experiments. the differences between groups were assessed by student's t test. a p value < . was considered statistically significant. all statistical analyses were performed using spss. additional file : figure s . modulation of bip and chop expression in the persistently jev-infected cell clones. protein samples were collected from bhk- cells infected with jev at an moi of at to hr p.i., and from persistently jev-infected (pi) cell clones cbs - and cbs - . cell lysates were analyzed by western blotting for chop, bip, jev ns , and the internal control β-actin. band intensities for bip and ns were determined by densitometry and normalized to those for β-actin. submit your next manuscript to biomed central and take full advantage of: persistent infection of tissue culture cells by rna viruses strategies of virus persistence molecular mechanisms of measles virus persistence molecular mechanisms of persistent infection by reovirus endoplasmic reticulum stress in disease pathogenesis the unfolded protein response: from stress pathway to homeostatic regulation modulating stress responses by the uprosome: a matter of life and death the endoplasmic reticulum and the unfolded protein response identification of novel stress-induced genes downstream of chop roles of chop/gadd in endoplasmic reticulum stress mechanisms of er stress-induced apoptosis in atherosclerosis integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress viruses, endoplasmic reticulum stress, and interferon responses west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion dengue virus modulates the unfolded protein response in a time-dependent manner dengue virus serotype infection specifies the activation of the unfolded protein response hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response hepatitis c virus ns protein triggers endoplasmic reticulum stress and suppresses its own viral replication overview: japanese encephalitis persistence of japanese encephalitis virus in the human nervous system persistent infection of cultured mammalian cells by japanese encephalitis virus continuous mouse brain cell lines chronically infected with japanese encephalitis virus clonal analysis of mammalian cell cultures persistently infected with japanese encephalitis virus persistent infection of porcine kidney cells with japanese encephalitis virus antiapoptotic but not antiviral function of human bcl- assists establishment of japanese encephalitis virus persistence in cultured cells integrative effect of defective interfering rna accumulation and helper virus attenuation is responsible for the persistent infection of japanese encephalitis virus in bhk- cells characterization of homologous defective interfering rna during persistent infection of vero cells with japanese encephalitis virus persistence, latency and reactivation of japanese encephalitis virus infection in mice defective interfering rnas of japanese encephalitis virus found in mosquito cells and correlation with persistent infection japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response effect of enforced expression of human bcl- on japanese encephalitis virus-induced apoptosis in cultured cells stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy er stress, autophagy, and rna viruses translational control in the endoplasmic reticulum stress response upregulation of chop/gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway bip overexpression, but not chop inhibition, attenuates fatty-acid-induced endoplasmic reticulum stress and apoptosis in hepg liver cells chop is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum chop induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum we would like to thank professor radhakrishnan padmanabhan (georgetown university, usa) for providing flavivirus ns -specific antibody. the authors declare that they have no competing interests. key: cord- -nvyknvc authors: delangue, julie; roca sanchez, yelin; piorkowski, géraldine; bessaud, maël; baronti, cécile; thirion-perrier, laurence; mafayle, roxana loayza; ardaya, cinthia avila; aguilera, gabriela añez; guzman, jimmy revollo; riera, javier lora; de lamballerie, xavier title: viral aetiology influenza like illnesses in santa cruz, bolivia ( – ) date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: nvyknvc background: acute respiratory infections represent a serious public health issue worldwide but virological aetiologies of influenza like illnesses (ilis) remain largely unknown in developing countries. this study represents the first attempt to characterise viral aetiologies of ilis in bolivia. methods: it was performed in santa cruz city from january to september , based on naso-pharyngeal swabs collected in a national reference laboratory and real-time pcr techniques, viral cultures and phylogenetic analyses. results: . % of samples were positive for at least one virus with influenza viruses (flu a: ~ %; flu b: ~ %), rhinoviruses (~ %), coronaviruses (~ %) and hrsv (~ %) being the most frequently identified. the pattern of viral infections varied according to age groups. the elucidation rate was the highest (> %) amongst patients under yo and the lowest (< %) amongst patients ≥ yo. nearly % of samples showed dual viral infections. epidemiological peaks were associated with a predominant virus but generally included - % of infections by different viruses. unexpectedly, the frequency of influenza in the – yo population was very low and a complete hrsv eclipse occurred in . genetic analyses indicated that distinct evolutionary lineages of flu a(h n )pdm , flu a/h n and flu b have co-circulated in bolivia in the study period, originating from central and north america, europe, asia and australia. conclusion: our results emphasise the requirement for a reinforced epidemiological and genetic follow-up of influenza and other ilis in bolivia to further inform the preparation of vaccines used in the region, guide vaccination campaigns and improve the medical management of patients. acute respiratory tract infections (aris) represent a leading cause of human acute illnesses worldwide and an important contributing factor of childhood morbidity and mortality, especially under the age of [ ] [ ] [ ] . besides the issue of bacterial pneumonia, which represents a frequent cause of mortality in paediatric populations of developing countries, there is an increasing interest in the epidemiology and characterization of influenza like illnesses (ilis), which are predominantly of viral aetiology [ ] . first, ilis are responsible for a large number of cases in all age groups (with a specifically high frequency in children) and despite their frequent association with a mild clinical presentation, their medical and socio-economical impact is enormous [ ] . second, a proportion of ilis is associated with severe clinical evolution [ ] . in adults, viruses are the putative causative agents in a third of cases of community-acquired pneumonia, in particular influenza viruses, rhinoviruses, and coronaviruses. dual viral infections are common, and a large proportion of children have evidence of viral-bacterial co-infection [ ] . deciphering the epidemiological and pathophysiological links between viral primary infection and secondary bacterial super-infection and pneumonia currently represents a major challenge for improving the prevention and medical management of pneumonia. despite their importance in terms of morbidity as well as infant mortality, microbiological aetiologies of aris represent a complex, which has yet to be fully characterized in developed countries and remains largely unknown in developing countries. despite recent improvements due to prospective studies launched during or following the influenza a(h n )pdm in peru and ecuador [ , ] , the information about ilis in the tropical hispanic regions of south america is scarce and the epidemiology remains insufficiently characterised. here, we have performed the first study of ilis in bolivia, which covers years from january to september . the viral aetiology of infections was investigated based on naso-pharyngeal swabs collected in a national reference laboratory in the city of santa cruz de la sierra and real time pcr molecular biology techniques, viral cultures and phylogenetic analyses. the number of samples that could be analysed each year at centro nacional de enfermedades tropicales, cenetrop was determined according to logistical considerations ( in , in and in ; total = ). we did not attempt to build a representative sample of specimens received at cenetrop (supposedly poorly representative of ilis in the general population of santa cruz), but rather examined a similar number of samples in each age group to allow a comprehensive aetiological analysis in all age classes. our final sample has equivalent numbers of swabs in the - , - , - and - age classes ( to per class) but slightly lower numbers in the older classes ( - : ; - : ; ≥ : ) due to the limited number of samples received at cenetrop for these age groups. as expected, it under-represents the - years old (yo) age group (which includes~ % of eligible samples received), and over-represents the - , - , - and ≥ yo age groups (additional file ). the general characteristics and distribution of cases are presented in table . the gender ratio (m/f) is . and the history of influenza vaccination clearly shows that a limited proportion of the population received influenza vaccines, in particular in the elderly ( . % have received at least one dose of influenza vaccine in their life in the ≥ yo age group). of the samples tested, ( . %) were positive for at least one virus (table ) with influenza viruses (flu a:~ %; flu b:~ %), rhinoviruses (hrv) (~ %), coronaviruses e &oc (hcov e &oc ) (~ %) and respiratory syncytial virus hrsv (~ %) being the most frequently identified. the pattern of viral infections varied according to age groups ( figure a ). the elucidation rate was the highest (> %) in samples from patients under the age of yo and the lowest (< %) in patients ≥ yo (figures a and b) . since all samples originated from symptomatic patients, this may be due either to the higher prevalence of the viruses tested or to the higher level of viral shedding in children. the - yo age group displayed specific epidemiological characteristics with the majority of hrsv infections (infection rate: . %) and a remarkably low proportion of influenza infections: < . % compared with~ % in the - yo age group,~ . % in the - yo age classes and~ % in those ≥ yo. adenoviruses were most frequently identified in children under the age of , bocaviruses in patients under the age of and hcov e & oc , although present in all age groups, were more prevalent in patients ≥ yo. interestingly, metapneumoviruses (hmpv) were detected in both children and adults with the most elevated rate of infection in the ≥ yo age group (~ %) and enteroviruses/parechoviruses were also found in both children and adults. rhinoviruses were identified in all age groups (up to~ % in patient over years of age). nearly % of samples showed dual viral infections ( table ): ( . %) of the influenza a viruses, ( . %) of the rhinoviruses and ( . %) of the hcov e &oc infections detected were diagnosed in a situation of co-infection. co-infections were specifically frequent in the case of adenoviruses ( . %). despite a high number of influenza b cases ( ) no co-infection was noted. the rate of dual infections was high for bocaviruses and enteroviruses/parechoviruses (≥ %) and low for metapneumoviruses and parainfluenzaviruses (< %) but this was based on a limited number of cases. the median age was not different in patients with single or dual infections. the curve of positive samples followed that of samples tested ( figure b ) and that of samples received at cenetrop (figure a ). peaks of detection matched peaks of reception of samples, which globally followed the seasonal subtropical pattern of ilis observed in bolivia, with one peak during the austral autumn and a second during the austral winter season. in , a great number of samples could be studied ( ) allowing to precisely follow several peaks of activity corresponding to a mosaic of viruses including flu b, hrsv, hrv, flu a a/h n and a (h n )pdm and hcov e &oc . in and , the number of samples studied annually was lower ( and , respectively), but still sufficient to elucidate the main peaks of activity with a major incidence of flu a (h n )pdm in and flu b in , co-circulating with a variety of other viruses (see figure c ). epidemiological peaks were generally associated with a predomin- this is summarised in figure c that shows a clear epidemiological evolution. in , following the flu a(h n )pdm pandemic wave, influenza a infections were for the most part due to flu a/h n and associated to a large number of flu b and rhinovirus infections and a variety of less prevalent viruses. by contrast, year was characterised by the massive come back of flu a(h n )pdm , the low number of flu b, the emergence of a large number of hcov e &oc infections and the absence of hrsv infections. in , the situation changed again with a large number of flu b and hrsv infections and the persistence of a significant proportion of rhinovirus and flu a(h n )pdm infections. altogether, this provides a striking picture of a moving infection pattern, which is presumably modelled by the herd immunity in the most epidemiologically susceptible populations. the complete disappearance of hrsv infections in represents a specific case that was never encountered by the authors in europe. cell culture isolates of influenza a/h n (n = ), influenza a(h n )pdm (n = ) and influenza b (n = ) viruses were obtained. eight-segment genomic sequences were produced for flu a/h n isolates (genbank accession numbers kf -kf ) and flu a/h n isolates (genbank accession numbers kf -kf ). partial sequences (segments ha, na, ns, m) were produced for flu b isolates (genbank accession numbers kf -kf ). the corresponding sequences were used for further phylogenetic analyses. influenza b viruses are divided into two antigenic lineages (victoria/ and yamagata/ ) since . both of them frequently co-circulate during epidemic periods [ ] and segment reassortment between strains of both lineages is frequent, making it difficult to produce robust complete-genome phylogenetic analyses. here, evolutionary trees were reconstructed from the alignment of ha sequences and showed that ten out these data suggest that variants belonging to distinct evolutionary clusters have circulated simultaneously in south america during the and influenza seasons. this information is relevant for optimising the formula of the seasonal influenza vaccine in order to provide efficient protection [ ] . influenza a(h n )pdm figure ) . altogether, the main information arising is that several evolutionary lineages co-circulated in bolivia during the h n pandemic wave. this study represents the first attempt to characterise the viral aetiology of ilis in bolivia and the first in south america in the post-influenza a(h n )pdm period. it was not designed for providing a representative picture of the general bolivian population: (i) samples originated from the region of santa cruz only (this item is further discusses below); (ii) samples sent to cenetrop were not expected to faithfully represent ilis in the region of santa cruz, with obvious recruitment biases such as clinical severity or access to public medical care; (iii) sampling was performed with the objective of obtaining equivalent numbers of cases in all age classes. the information obtained should therefore be analysed with precautions. firstly, the great year-to-year variation of infection epidemiological patterns is a reminder that the epidemiology of ilis should be studied over long periods of time. each of the three years studied here would have provided, if analysed separately, a partial view of the situation and would have lead to erroneous conclusions (e.g., the absence of hrsv infections in bolivian children extrapolated from data). the analysis of a -year period is expected to smooth such epidemiological jolts but an extended observation period would have undoubtedly revealed more information about the viruses that circulated at low rates in the period studied (e.g., parechoviruses, parainfluenzaviruses). secondly, bolivia includes territories with diverse geographical, climatic and ecological characteristics. santa cruz is located in an eastern low-altitude area with a humid tropical climate whilst la paz is in a western andean high-altitude area with a colder and dryer climate [ ] . this may explain important epidemiological differences observed between both cities, as exemplified by the case of influenza a(h n )pdm : in , we observed a very limited number of flu a(h n )pdm infections in santa cruz whilst the paho surveillance system reported an intense circulation of the virus in la paz during the first months of the year (paho surveillance website: http://ais.paho. org/phip/viz/ed_flu.asp); in , a large outbreak occurred in santa cruz (see figures and ) , whilst only a few cases were observed in la paz; in , an important outbreak was observed in la paz, but not in santa cruz. this clearly indicates that epidemiological data collected in santa cruz cannot be extrapolated easily to the andean regions of bolivia. divergent influenza epidemiological patterns according to geographical distribution were finely documented in a previous peruvian study [ ] . despite the fact that the local epidemiology of aris is modelled by herd immunity and environmental factors, the global picture of aris that arises from our study conforms with that observed in different regions of the world. in particular, the choice of the pathogens tested seems to be relevant since (i) the rate of elucidation is close to what is observed in france, using the same molecular assays (authors' personal data), (ii) peaks of detection match the ili epidemiological peaks and (iii) in previous studies performed in brazil, the different viruses identified in the current study were detected, confirming their circulation in the region [ ] [ ] [ ] [ ] [ ] . the distribution of cases in age groups is also essentially similar with what was reported elsewhere, e.g., in ecuador [ ] . the most surprising items, which deserve investigations on an extended period of time, were the unusually low frequency of influenza infections in the - yo age group and the complete rsv eclipse in . however, precise comparison with data collected in south america is most often difficult, e.g., with a molecular study in ecuador [ ] and a previous large study in peru [ ] (different periods investigated, viruses detected using immunofluorescence tests, parechoviruses, coronaviruses, metapneumoviruses and rhinoviruses not tested). the genetic characterisation of influenza strains indicates that viruses belonging to distinct evolutionary lineages of flu a(h n )pdm , flu a/h n and flu b have co-circulated in bolivia. interestingly, an increasing number of influenza sequences has been made available since and phylogenetic reconstructions revealed, on the one hand, the existence of south american evolutionary clusters of influenza strains, and on the other hand a complex epidemiological relationship with the rest of the world. trees suggested the circulation in bolivia during the study period of viruses originating from central and north america, europe, asia and australia. this study provided the first systematic and broadspectrum information regarding the viral aetiology of ilis in bolivia. a continued effort is needed to accurately characterise the epidemiology of ilis in the different regions of the country to guide vaccination campaigns for at-risk populations and improve the medical management of patients. in accordance with previous investigations performed in peru [ ] , our results emphasise the requirement for a reinforced genetic follow-up of influenza strains circulating in south america to further inform the preparation of vaccines used in the region. the recruitment of cases was operated following the broad spectrum who case definition of ilis: anybody with fever of . °c or higher, associated with one respiratory symptom was identified as ili infection and eligible for investigation. we used naso-pharyngeal swabs sampled in hospitals and health centres in the region of santa cruz and sent to the cenetrop (centro nacional de enfermedades tropicales, santa cruz, bolivia) for diagnosis from january to september . all specimens had been sampled between day and day following clinical onset, sent to the laboratory at room temperature using a universal transport media and kept at − °c upon reception. all patients or their legal representative gave oral consent for the shipment of samples to cenetrop and the detection of respiratory pathogens. to provide a preliminary report of viral aetiologies in all age groups (i.e., information that may be translated easily for public health purposes), a random selection of an equivalent number of samples was operated in age groups ( - , - , - , - , - , - and ≥ years of age) for each year of the study. aetiological identification was performed using taqman probe-based real-time pcr and rt-pcr protocols. samples were spiked by ms and t phages, used as internal controls [ ] . rna [ ] ), respiratory syncytial viruses a and b (hrsv a, hrsv b, [ ] ), coronaviruses e &oc (hcov e &oc , [ ] ), rhinovirus (hrv, [ ] , adenoviruses (hadv, [ ] ), enteroviruses & parechoviruses (hevs, hpev, [ ] ), human metapneumoviruses a and b (hmpv, [ ] ), human bocaviruses (hbov, [ ] ). samples providing a strong positive real-time pcr result (ct < ) for influenza a or b viruses were used for virus isolation using mdck cells and a standard isolation protocol (additional file ). in addition to strains isolated in the course of the current study, influenza a(h n )pdm virus strains isolated at cenetrop in the june-august period were sequenced to reinforce phylogenetic analyses (see below). sequencing μl of clarified supernatant from influenza a or b positive cell cultures were extracted using the ez ® qiagen instrument and the ez ® virus mini kit v . without rna carrier (qiagen). all segments of influenza a virus were amplified in a single reaction using the superscript® iii one-step rt-pcr system with the plat-inum® taq high fidelity kit and the universal primers univ and univ [ ] . to obtain influenza b virus sequences, we used a similar protocol and one set of primers (bm-ns- and bm-ns- ) to amplify the ha, na and ns segments [ ] and another set of newly designed primers (m- efor: ag aagyasagcattttcttgtga and m- erev: taaa cacccacatyccaaacgt) to amplify the m segment. pcr products were purified (nucleofast pcr plate, machrey nagel) and sequenced using the ngs ion torrent technology: briefly, dna concentrations were adjusted to nm (qubit® dsdna br assay kit and qubit® . fluorometer) and a library of barcoded - nt fragments was built using the diagenode bioruptor ucd- , the xpress™ barcode adapters kit and the ab library builder™ (life technologies). clonal amplification was performed using the ion pgm™ template ot kit and the ion onetouch™ instrument (both from life technologies). final sequencing was performed using the personal genome machine® (pgm™) system, the ion pgm™ sequencing kit v and the ion ™ chip kit (life technologies). estimates of world-wide distribution of child deaths from acute respiratory infections who estimates of the causes of death in children global burden of acute respiratory infections in children: implications for interventions viral pneumonia rhinoviruses: markers of, or causative for, recurrent wheeze and asthma? child health epidemiology reference group of who and unicef: global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since epidemiology of influenza-like illness in the amazon basin of peru sentinel surveillance of influenza-like-illness in two cities of the tropical country of ecuador the evolutionary dynamics of human influenza b virus writing committee of the world health organization consultation on southern hemisphere influenza vaccine composition for : who recommendations for the viruses to be used in the southern hemisphere influenza vaccine: epidemiology, antigenic and genetic characteristics of influenza a(h n )pdm , a(h n ) and b influenza viruses collected from global influenza seasonality: reconciling patterns across temperate and tropical regions acute respiratory infection and influenza-like illness viral etiologies in brazilian adults sentinel surveillance of influenza and other respiratory viruses, brazil epidemiological and clinical features of human coronavirus infections among different subsets of patients acute respiratory viral infections in children in rio de janeiro and teresopolis full genome sequence analysis of parechoviruses from brazil reveals geographical patterns in the evolution of non-structural genes and intratypic recombination in the capsid region rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than , routine pcr tests a simple method for molecular detection of swine-origin and human-origin influenza a virus comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr rapid simultaneous detection of enterovirus and parechovirus rnas in clinical samples by one-step real-time reverse transcription-pcr assay real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages real-time pcr assays for detection of bocavirus in human specimens full genomic amplification and subtyping of influenza a virus using a single set of universal primers screening of the high yield influenza b virus on mdck cell and cloning of its whole genome muscle: multiple sequence alignment with high accuracy and high throughput mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods viral aetiology influenza like illnesses in to produce consensus sequences for each isolated strains, reads obtained from using the ion torrent protocol were processed using the clc genomics workbench software . (clcbio, aarhu, denmark) and a home developed program and then mapped onto concatenated reference sequences.in the case of a/h n ( ) phylogenies, concatenated coding sequences of the eight segments from the complete genomic sequences produced as reported above from bolivian strains were aligned with complete genomic sequences retrieved from databases (total: , genomic sequences). in the case of a/h n and influenza b phylogenies we collected complete genomic sequences from databases but used only the ha gene sequences to produce alignments (a/h n : , sequences; influenza b: sequences). alignments were conducted using the muscle programme [ ] , implemented in the influenza research database (multiple sequence alignment: http:// www.fludb.org/brc/) and subsequently used to perform phylogenetic analyses using the mega . programme [ ] , the jukes cantor algorithm for distance calculation and the neighbour joining method for tree building. additional file : distribution of the eligible and studied populations in age groups.additional file : influenza b phylogeny cluster . this is a magnification of influenza b phylogeny cluster ( figure ). legend shape represents the year of strain isolation: square for ; circle for ; triangle with the tip up for , triangle with the tip down for ; rhombus for < . the colour represents the geographical origin: light blue for bolivian strains from this study; pink for bolivian strains from databases; dark blue for south american strains from databases. the authors declare that they have no competing interests.authors' contributions jd: participate on sampling samples molecular biology, culture and sequencing parts and draft of paper; yrs: participate to contributions to conception and design of project. gp: have made substantial contributions to conception and design of influenza sequencing participated in the sequence treatment alignment. mb: helps analysis and interpretation of data. cb; have made substantial contributions to conception and design of influenza sequencing. ltp; have been involved in drafting the manuscript. rlm: participate on molecular biology and viral culture. caa: participate on sampling samples molecular biology. gaa: participate on sampling samples molecular biology. jrg; participate to contributions to conception and design of project. jlr: participate to contributions to conception and design of project. xdl: have given correction and final approval of the version to be published. all authors read and approved the final manuscript. submit your next manuscript to biomed central and take full advantage of: key: cord- -gc o ou authors: kim, heui man; lee, namjoo; kim, mi-seon; kang, chun; chung, yoon-seok title: characterization of neuraminidase inhibitor-resistant influenza virus isolates from immunocompromised patients in the republic of korea date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: gc o ou background: the emergence of influenza viruses resistant to anti-influenza drugs is a threat to global public health. the korea centers for disease control and prevention operates the korea influenza and respiratory viruses surveillance system (kinress) to monitor epidemics of influenza and severe acute respiratory infection (sari) to identify mutated influenza viruses affecting drug resistance, pathogenesis, and transmission. methods: oropharyngeal swab samples were collected from kinress and sari during the – season. the specimens confirmed influenza virus using real-time rt-pcr on inoculated mdck cells. ha and na sequences of the influenza viruses were analyzed for phylogeny and mutations. neuraminidase inhibition and hemagglutination inhibition assays were utilized to characterize the isolates. results: two a(h n )pdm isolates harboring an h y substitution in the neuraminidase sequence were detected in patients with acute hematologic cancer. they had prolonged respiratory symptoms, with the virus present in the respiratory tract despite oseltamivir and peramivir treatment. through the neuraminidase inhibition assay, both viruses were found to be resistant to oseltamivir and peramivir, but not to zanamivir. although hemagglutinin and neuraminidase phylogenetic analyses suggested that the a(h n )pdm isolates were not identical, their antigenicity was similar to that of the – influenza vaccine virus. conclusions: our data indicate the utility of monitoring influenza-infected immunocompromised patients in general hospitals for the early detection of emerging neuraminidase inhibitor-resistant viruses and maintaining continuous laboratory surveillance of patients with influenza-like illness in sentinel clinics to monitor the spread of such new variants. finally, characterization of the virus can inform the risk assessment for future epidemics and pandemics caused by drug-resistant influenza viruses. neuraminidase inhibitors (nais) are globally utilized for the treatment and prevention of influenza types a and b infection [ ] , which are stockpiled by some countries, including the republic of korea, against unexpected pandemics. nais suppress the action of neuraminidase (na) on the surface of the virus to prevent the spread of progeny virus from infected cells [ ] . oseltamivir and peramivir, the most commonly used drugs for patients with influenza-like illnesses in the republic of korea, help to relieve clinical symptoms within days of symptom manifestation and shorten the virus-release period in the respiratory tract [ ] . baloxavir, a cap-dependent endonuclease inhibitor, was also recently licensed in the united states, following japan [ ] . however, since most recently detected type a influenza viruses harbor the resistance variation s n in the matrix gene, the m proton-channel blockers amantadine and rimantadine are no longer clinically applied [ ] . the korea centers for diseases control and prevention (kcdc) has operated the korea influenza and respiratory viruses surveillance system (kinress) to monitor epidemic features of influenza viruses and analyze virus characteristics, including drug resistance, since . the kcdc carries out diagnostic testing for influenza viruses in respiratory specimens requested by general hospitals as a national standard laboratory. if a drug resistance mutation is found in the na gene, the kinress attempts to isolate the virus and perform phenotypic analysis (na inhibition assay). the typical drug-resistance substitutions in na include h y, e d/g, and q r for a(h n )pdm ; e v, d g/v/d, r k, e d, r k, and n s for a (h n ); and g e, e a/d, h y, and r k for b virus [ ] , although additional single and combination mutations may also result in nai drug resistance (world health organization (who)) [ ] . flusurver provides genetic analysis tools for screening drug-resistance and variant mutations to facilitate genetic characterization of viruses [ ] . fluorescence-based na inhibition assays using the munana substrate have been conducted to confirm drug-resistant virus phenotyping [ ] . nai-resistant viruses were identified in cases in which influenza virus was continuously detected following nai treatment of hospitalized, immunosuppressed patients, rather than in clinical outpatients [ ] . here, drug-resistant a(h n )pdm viruses were detected via the kinress in patients with acute hematologic cancer not exhibiting recovery despite oseltamivir and peramivir administration; these were characterized genetically and antigenically following isolation. oropharyngeal swab samples were collected from patients with influenza-like illness through the kinress and processed in the national standard laboratory. swabs were stored in viral transport media (bd, san jose, ca, usa) until further analysis. viral rna was extracted from μl of sample medium using the qiaamp viral rna mini kit (qiagen, hilden, germany), according to the manufacturer's instructions. rt-pcr was carried out using the agpath id one-step rt-pcr system (applied biosystems, foster city, ca, usa) [ ] . table lists multiplex (a/b/ipc and h /h / ipc) primer and taq-man probe sets. the -μl reaction mixture contained μl of rna template, nm primers, nm probe, μl of × reaction buffer, . μl of enzyme mixture, and rnase-free water. rt-pcr was performed on the abi fast instrument (applied biosystems) with thermocycler conditions for reverse transcription ( °c, min), hot start dna taq polymerase activation ( °c, min), followed by cycles of denaturation ( °c, s) and annealing/extension ( °c, s). data acquisition and analysis of the real-time pcr assay were performed using sds software version . (applied biosystems). madin-darby canine kidney (mdck, american type culture collection, manassas, va, usa) cells were seeded at /ml in t flasks with dulbecco's modified eagle medium (hyclone, logan, ut, usa) containing % fetal bovine serum (hyclone) and % penicillinstreptomycin (sigma, st. louis, mo, usa) in a final volume of ml and incubated at °c in a humidified atmosphere with % co for h. when the cells reached % confluence, the culture medium was discarded, and the cells were washed three times with warm phosphatebuffered saline. then, . ml of clinical specimen was added into a flask to allow the inoculum to adsorb ( °c, min). medium ( ml) containing μl/ml tpck-trypsin was added to the flask. the culture was observed daily for cytopathic and morphological changes using an inverted light microscope. the culture supernatant was harvested when - % of the infected cells showed cytopathic effect and stored at − °c [ ] . finally, a/korea/s / and a/korea/s / a(h n )pdm viruses were isolated. viral rna was extracted from oropharyngeal swab samples and their isolates using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. reverse transcription was performed to obtain cdna using the u primer, then the ha and na genes were amplified with specific primers [ ] . the pcr products were purified using the qiaquick™ pcr purification kit (qiagen) according to the manufacturer's instructions and subjected to direct sequencing using the big dye terminator v. . cycle sequencing ready reaction kit (applied biosystems) together with primers for each pcr fragment ( (gisaid: epi and epi ) and na (gisaid: epi and epi ) sequences were obtained from the a(h n )pdm viruses. genetic screening for drug resistance in the na gene and mutational analysis of the ha and na genes were conducted using flusurver (https://flusurver.bii.a-star. edu.sg/), last updated on july , . phylogenies were reconstructed using the neighbor-joining method and bootstrapped times with mega x . using haor na-aligned dna sequences of the a(h n )pdm reference vaccine [ ] and other isolates from the republic of korea. a fluorescence-based neuraminidase inhibition assay was conducted using the na-fluor™ influenza neuraminidase assay kit (applied biosystems), according to the manufacturer's instructions. the susceptibility of influenza viruses to nai was characterized using oseltamivir, zanamivir, and peramivir at concentrations that inhibited the na activity by % (ic ) as described previously [ ] . as defined by the who, influenza a viruses with normal, reduced, and highly reduced na activity inhibition exhibit a < -fold, -to -fold, and ≥ -fold increase in ic , respectively; the last are considered clinically resistant [ ] . assays were performed according to standard methods [ ] . four ha units/ μl of a(h n )pdm virus were tested using . % suspensions of turkey red blood cells. hi titers were reciprocals of the highest dilutions of sera that inhibited hemagglutination. post-infection ferret antisera against - and - vaccine viruses were treated with receptor-destroying enzyme (denka seiken, tokyo, japan). (fig. ) . table shows the amino-acid substitutions in the ha gene compared with a/brisbane/ / and their reported effects. a/korea/s / and a/korea/s / viruses exhibited significantly reduced inhibition by oseltamivir and peramivir with > ic fold-changes relative to drugsensitive virus, but were normally susceptible to zanamivir (table ). however, every isolate collected from the kin-ress in - was susceptible to all three drugs. the two viruses were well inhibited by antisera from ferrets immunized with a/michigan/ / and a/brisbane/ / , which are northern-hemisphere influenza a(h n )pdm isolates collected in - and - , respectively, with less than two-fold reduced hi titer relative to homologous virus (table ) . the nai-resistant a(h n ) virus harboring the na gene h y variation was first identified in , and was frequently identified worldwide in - [ ] . however, since the a(h n )pdm pandemic, it occurred in < % of a(h n )pdm , mainly reported in patients with impaired immune systems due to immunosuppressive therapies for conditions including cancer and organ transplants [ , ] . notably, such immunocompromised patients, when infected with influenza virus, could shed virus over a prolonged period, likely causing increased transmission. moreover, as they shed drug-resistant virus while maintaining transmissibility, these patients may constitute a source for spreading drug-resistant influenza in the community [ ] . although nai-resistant virus has been rarely observed since , the kcdc, through the kinress, identified oseltamivir and peramivir-resistant a(h n )pdm viruses harboring the h y na variation in patients with acute lymphoblastic leukemia and relapsed lymphoma hospitalized in the same general hospital, with both exhibiting prolonged virus excretion. although there was no history of the two patients occupying the same ward, we were concerned about potential nai-resistant virus transmission. the phylogenetic analysis showed that the two isolated a(h n )pdm viruses were genetically distinct. moreover, there were no additional reports of similar viruses in that hospital. furthermore, van der vries et al. [ ] demonstrated that a(h n )pdm virus-infected immunocompromised ferrets exhibited prolonged virus replication despite antiviral therapy, along with the h y substitution observed in the virus population from day onwards only in ferrets that received oseltamivir. these results suggest that the h y substitution emerges rapidly in immunocompromised hosts under continuous antiviral selective pressure. mutations suspected to represent antigenic drift in immunocompromised patients were more likely to be observed in na than ha, albeit at a very low frequency (around %). however, the na mutations did not affect the na h y substitution and some ha or na substitutions in drug-resistant a(h n )pdm viruses isolated from immunocompromised patients influence both antigenicity and nai resistance. the viruses showed highly reduced inhibition, with over -fold increases in the ic for oseltamivir and peramivir, but not zanamivir. thus, zanamivir might be an option for immunocompromised patients when oseltamivir and peramivir are not effective. these findings also indicate the necessity of monitoring for nai-resistant viruses in outpatients visiting clinics and in-patients, particularly for immunocompromised patients (e.g., with severe acute respiratory syndromes in general hospitals). additionally, virus characterization can facilitate the risk assessment of nai viruses. effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analyses of randomized controlled trials influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir viruses baloxavir: first global approval incidence of antiviral drug resistance markers among human influenza a viruses in the eastern mediterranean region rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus a (h n ), a (h n ) , and a (h n ) subtypes by melting point analysis summary table of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (nai) neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods prolonged shedding of multidrugresistant influenza a virus in an immunocompromised patient development and evaluation of multiplex real-time rt-pcr assays for seasonal h pdm and avian a/h influenza viruses detection susceptibility of influenza a (h n )/pdm , seasonal a (h n ) and b viruses to oseltamivir in guangdong universal primer set for the full-length amplification of all influenza a viruses european centre for disease prevention and control. influenza virus characterization, summary europe global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals summary of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (nai) world health organization. manual for the laboratory diagnosis and virological surveillance of influenza neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts development of oseltamivir and zanamivir resistance in influenza a (h n ) pdm virus prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets hemagglutinin receptor specificity and structural analyses of respiratory droplet-transmissible h n viruses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the national influenza surveillance participating institutes. conception and design: chun kang, yoon-seok chung. acquisition of data: namjoo lee, mi-seon kim. analysis and interpretation: heui man kim, yoon- seok chung. writing, review and revision of the manuscript: heui man kim. study supervision: chun kang, yoon-seok chung. all authors read and approved the final manuscript. this study was supported by intramural funding from korea centers for disease control and prevention ( - ). the data supporting the conclusions of this article are included within the article.ethics approval and consent to participate all experimental procedures were approved by the institutional review boards of korea centers for disease control and prevention (ethics number: - - -p-a). not applicable. the authors declare that they have no competing interests.received: march accepted: june key: cord- -frej qmb authors: nakouné, emmanuel; tricou, vianney; manirakiza, alexandre; komoyo, francis; selekon, benjamin; gody, jean chrysostome; victoir, kathleen; buchy, philippe; kazanji, mirdad title: first introduction of pandemic influenza a/h n and detection of respiratory viruses in pediatric patients in central african republic date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: frej qmb background: acute viral respiratory illnesses in children in sub-saharan africa have received relatively little attention, although they are much more frequent causes of morbidity and mortality than in developed countries. active surveillance is essential to identify the causative agents and to improve clinical management, especially in the context of possible circulation of pandemic viruses. findings: a prospective study was conducted in the central african republic (car) between january and december among infants and children aged – years attending sentinel sites for influenza-like illness or acute respiratory illness. nasopharyngeal swabs were collected, and one-step real-time and multiplex reverse transcription-polymerase chain reaction were used to detect respiratory viruses. respiratory viruses were detected in of the ( . %) nasopharyngeal samples: ( . %) contained influenza viruses ( ( . %) had pandemic influenza a/h n virus and ( . %) had influenza b viruses), ( . %) contained parainfluenza viruses types and and ( . %) contained human respiratory syncytial virus. most cases were detected during the rainy season in the car. analysis of the amplicon sequences confirmed the identity of each detected virus. conclusions: the influenza surveillance system in the car has provided valuable data on the seasonality of influenza and the circulation of other respiratory viruses. our network could therefore play a valuable role in the prevention and control of influenza epidemics in the car. although acute respiratory illness is a major cause of morbidity and mortality among children in sub-saharan africa, it has received relatively little attention [ ] . this is unfortunate, as underlying diseases such as aids, malaria and tuberculosis, which are highly prevalent in the region, can worsen such illnesses [ ] . the respiratory viruses known to cause acute illness include human respiratory syncytial virus (hrsv), human parainfluenza virus (piv), human metapneumovirus and influenza viruses [ ] [ ] [ ] . until recently, the burden of influenza and influenza-like illness in africa was considered to be negligible [ ] , mainly because of the lack of confirmation assays. reports from cameroon and senegal, however, show that influenza viruses are actively circulating and may be causing regular epidemics [ , ] . a clear picture of the contribution of each pathogen to acute respiratory illness is needed in order to improve prevention and clinical management and consequently to reduce the burden of disease. the emergence of the novel influenza a/h n of swine origin in mexico in april and its rapid spread worldwide, causing a global pandemic, led the health authorities of the central african republic (car) to collaborate with the world health organization in strengthening biological surveillance of acute respiratory illness. the aim of the study reported here was to determine the circulation of pandemic influenza a/h n virus (h n pdm ) by molecular methods and to identify the causative viruses, the incidence and the clinical features of acute respiratory illness among infants and young children at sentinel sites in bangui and three rural areas. all infants and children aged between - years who attended sentinel sites in bangui and three rural areas ( figure ) for influenza-like illness (ili) or severe acute respiratory illness between january and december were included in the study (figure a ). the world health organization definitions were used for ili (sudden onset of fever of > °c and cough or sore throat in the absence of other diagnoses) and severe acute respiratory illness (ili symptoms and shortness of breath or difficulty in breathing and requiring hospital admission). the study protocol was approved by the national ethics committee of the car. individual written informed consent was sought from the parents or guardians of all participants. nasopharyngeal samples were collected from infants and children and within hrs at °c to the national influenza centre by using rayon-budded swabs with virus transport medium pre-impregnated sponge (virocult, medical wire & equipment, uk). rna was extracted with a qiamp rna mini kit (qiagen) according to the manufacturer's instructions. influenza a viruses were detected with a previously described assay targeting the conserved matrix gene for universal detection of these viruses [ ] , and h n pdm virus was identified with a specific one-step real-time reverse transcription-polymerase chain reaction (rt-pcr) assay (designed by the national influenza centre of northern france, institut pasteur, paris; primers and probe available upon request at grippe@pasteur.fr). all specimens were also tested for other respiratory viruses in two previously described multiplex semi-nested rt-pcr assays for detecting influenza a and b viruses, hrsv, human metapneumovirus and piv types , , and [ , ] . all assays were performed on an abi platform (applied biosystems, foster city, california, usa) with the superscript iii platinum one-step quantitative rt-pcr system (invitrogen, carlsbad, california, usa). a specimen was considered positive if the signal curve crossed the threshold line within cycles. the assay limit of detection for pandemic h n pdm influenza virus is of order of magnitude of copies/μl of initial sample [ ] . the assay limit of detection for influenza a and b viruses, hrsv, human metapneumovirus and piv- is of order of magnitude of copies/μl [ ] . for piv- , the assay limit of detection is of order of magnitude of copies copies/μl, and for piv- and − , copies/μl [ ] . after amplification, the pcr products were purified and sent to gatc biotech (konstanz, germany) for sequencing. student's t test and the pearson chi-squared test were used to assess intergroup differences. statistical analyses were performed with epiinfo software (v . . cdc). a test was considered significant when the p value was < . . the newly obtained sequences were analysed and compared with sequences available in genbank. of the patients included in the study, ( . %) tested positive for respiratory viruses (table ) . of these, five ( . %) were positive for h n pdm , ( . %) for influenza b, ( . %) for hrsv and ( . %) for piv- and − (table and figure b ). the average age of positive patients was months (range, month to years), with no difference in the age or gender distribution. all samples were negative for human metapneumovirus. the first case of h n pdm was detected in the car on july in a child aged months, followed by a second case on september in an infant aged months, and three cases were found in young children on , and october ( figure b ). of the five children, only one was admitted to intensive care for respiratory distress; no deaths were recorded. a -bp fragment of the haemagglutinin gene of the five h n pdm strains [accession numbers: cy , cy , cy , cy , cy ] showed high sequence similarity ( - %) to influenza a/california/ / (h n ) (data not shown). the specimens positive for influenza b represented . % of the influenza viruses detected. ten of the patients were hospitalized; one infant aged months died days after admission to intensive care with a clinical picture of severe acute respiratory illness. influenza b virus was implicated in respiratory infections hrsv was detected in patients (table ) with a median age of months, mainly in november ( figure b ). one infant aged months died day after admission to intensive care with a clinical picture of fever, cough, rhinitis, bronchiolitis, dyspnoea and myalgia. fever and rhinitis were recorded in of the patients, and all had cough. no viral co-infections were reported. analysis of the nucleotide sequences of six isolates [accession numbers: he , he , he , he , he , he ] showed sequence similarity to both genotypes a and b. piv- was detected in seven patients (median age, months; range, - months) and piv- in four infants (median age, months; range, - months) between april and november ( figure b ). respiratory viruses were found in . % of the collected samples, showing the presence of h n pdm infections in the car for the first time. we found that influenza b, piv- , piv- and hrsv were also involved in ili in the country. most of the cases were detected during the rainy season. h n pdm infection in the car was first described in july . in other african countries, the virus was shown to have been introduced by travellers [ ] [ ] [ ] , but its source in the car has not been elucidated. all four cases detected were indigenous, with no history of travel or contact with a person returning from a country with declared cases. extensive investigations of contacts of the confirmed cases did not reveal any other cases, suggesting low dissemination of h n pdm in the country. the clinical picture of h n pdm infection was similar to that of seasonal influenza circulating before the pandemic. this is in accordance with studies showing relatively low transmissibility and severity of h n pdm [ , ] . consequently, h n pdm did not appear to have had a significant public health impact in this area of the world. in the present study, influenza b virus was the most commonly detected respiratory virus (n= ), whereas in similar studies in africa hrsv was the most frequent causative virus of ili [ ] [ ] [ ] . influenza b virus caused respiratory infections throughout the year, with two peaks: at the beginning of the rainy season (june-july) and at the end of the rainy season (november). antigenically and genetically distinct lineages of influenza b virus, influenza b/victoria and b/yamagata viruses have circulated in the car [ ] . hrsv is a major cause of ili among infants and children worldwide [ ] and is the most frequently detected respiratory virus in both developed and developing countries [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, hrsv was found in only . % of the samples and represented . % of the viruses detected. the difference from other studies might be due to a different epidemiology of hrsv in the car, a landlocked country in central africa, or to different inclusion criteria or detection techniques. most of hrsv cases detected in our study occurred between november and february, corresponding to the dry season in the car. another interesting finding was the relatively low prevalence of respiratory viruses in the children with ili, which were present in only . % of samples; in similar studies, as many as % of samples contained respiratory viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the car, vaccines against streptococcus pneumoniae and haemophilus influenzae were introduced in the enlarged programme of vaccination only recently (h. influenzae in september and s. pneumoniae in july ). in a study in bangui in , these bacteria were found in hundreds of ill children aged less than years [ ] . it is therefore likely that these bacteria are still very common or even the main causes of respiratory infections in the car, as in other countries before the introduction of vaccines, when most severe respiratory infections were due to bacterial infections, and s. pneumoniae and h. influenzae were the commonest bacterial causes [ ] [ ] [ ] . this may explain, at least partly, the differences between our results and those of other studies. it is also possible that viruses other than these we looked for were involved, such as human rhinovirus, human bocavirus, human coronavirus or adenovirus. the main limitation of our study is the limited sample size, which prevented us from investigating associations between clinical outcome and viral etiology. another weakness is a bias toward younger patients, so that we could not assess whether a particular age group is at greater risk for a specific infection. owing to the design of the study, it is also difficult to determine prevalence from the results. therefore, further studies are needed to evaluate the burden of all respiratory viruses infections in the general population of the car. our study does highlight the importance of the clinical, epidemiological and virological network for influenza surveillance in the car [ , ] . we reported here the first data on the etiology of ili in the car. this will help central african clinicians to provide better care and treatment for patients presenting with ili, including better use of antibiotics. further studies with more patients are needed to confirm the burden of viral respiratory diseases in the car. collection of samples from healthy control children may also enable comment on virus detection and disease association. another suggestion for future studies is to collect data about underlying health status of children as risk factors such as hiv infection, malaria, malnutrition, etc. might have an impact on the acute respiratory infections [ , ] . the temporal patterns detected should be assessed over many years in order to identify long-term seasonal patterns. abbreviations car: central african republic; 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cheng, an-chun; wang, ming-shu; pan, kang-cheng; li, min; guo, yu-fei; li, chuan-feng; zhu, de-kang; chen, xiao-yue title: development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of goose parvovirus in vivo date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: yn r h background: goose parvovirus (gpv) is a dependovirus associated with latent infection and mortality in geese. currently, it severely affects geese production worldwide. the objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (pcr) (fq-pcr) assay for fast and accurate quantification of gpv dna in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of gpv in vivo. results: the detection limit of the assay was . × ( )standard dna copies, with a sensitivity of logs higher than that of the conventional gel-based pcr assay targeting the same gene. the real-time pcr was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. conclusion: the high sensitivity, specificity, simplicity, and reproducibility of the gpv fluorogenic pcr assay, combined with a high throughput, make this method suitable for a broad spectrum of gpv etiology-related applications. goose parvovirus (gpv) is the causative agent of gosling plague (gp), an acute, contagious, and fatal disease, which is also known as derzsy's disease [ ] . gpv has been formally classified as a member of the genus dependovirus in family parvoviridae [ ] . it was first described as a clinical entity by fang [ ] . it causes considerable economic losses, especially in countries with an industrialized goose pro-duction system, because the virus infection spreads rapidly worldwide causing high rate of morbidity and mortality [ , [ ] [ ] [ ] . regular methods for identifying gpv include agar-gel diffusion precipitin test, virus neutralization (vn) assay, and enzyme-linked immunosorbent assay (elisa) [ ] . however, these methods have certain limitations; they are tedi-ous and are not always reliable because of the requirement of specific-pathogen-free (spf) gosling embryos and standard positive anti-gpv serum [ , ] . recently, the highly conserved vp region of the gpv gene was cloned and sequenced and analyzed by qualitative polymerase chain reaction (pcr) assays [ ] [ ] [ ] [ ] . although qualitative pcr was useful for the diagnosis of gpv infection, it had some problems: it involved the electrophoresis and staining processes, which made the procedure lengthy, increased the risk of contamination, or rendered the method unsuitable for large-scale investigations [ ] [ ] [ ] . moreover, determination of the amount of virus in different tissues and cells was very useful for investigating the nosogenesis, virus replication, host-virus interactions, tropism, and effective for screening anti-viral drugs; all these factors could not be assessed by qualitative pcr [ , ] . in recent years, a method based on pcr with an automatic confirmation phase has been developed. this method, which is known as the fluorescent quantitative real-time pcr (fq-pcr), has been used widely to quantify the number of genomic copies of pathogenic microorganisms [ , ] . gpv detection by real-time pcr has only been reported by bi [ ] ; in that study, the method was not optimized and a fq-pcr standard curve was not generated. in this study, we reported the optimization of a fq-pcr assay to quantify gpv dna in vivo after experimental infection. the results of this study provide some interesting data that may be beneficial to understand the regular distribution pattern and nosogenesis of gpv in vivo in goslings. the concentration of standard pvp plasmid dna was μg/μl, and the a /a (ratio) was . ; the copy numbers of pvp plasmid dna were . × copies/ μl. after the optimization of fq-pcr, we selected the final concentrations of each primer as . μmol/l and that of probe as . μmol/l. the mgcl concentration was adjusted to mm to obtain optimal fq-pcr assay conditions. therefore, the optimized -μl fq-pcr reaction system for gpv detection was as follows: × pcr buffer, mmol/l mgcl , . mmol/l dntps, . μmol/l of each primer, . μmol/l of probe, u taq, and μl dna template. the optimized conventional pcr reaction system used in this study was as described by huang et al. [ ] : × pcr buffer, . mmol/l mgcl , . mmol/l dntps, . pmol/ l of each primer, . u taq, and μl dna template. the optimized annealing temperature was °c. the fq-pcr amplification curves and the corresponding fq-pcr standard curve ( figure ) were generated by employing the successively diluted known copy numbers of pvp for real-time pcr reaction under the optimized conditions. on the basis of the results of correlation coefficient ( . ) and pcr efficiency ( . %), it was confirmed that the standard curve and the established fq-pcr protocol were extremely effective. by using the following formula, we were able to quantify the amount of unknown samples: y = - . x + . (y = threshold cycle, x = log starting quantity). ten-fold dilutions of the pvp plasmid dna were tested by the established fq-pcr assay to evaluate the sensitivity of the system, and the detection limit was found to be . × copies/reaction. comparisons were made between the conventional pcr method and our established fq-pcr method using dilution series of pvp plasmid dna to calculate the end-point sensitivity of each assay. the results indicated that the established fq-pcr is approximately -times more sensitive than the conventional pcr method; the former method can detect pvp copies down to dilutions of . × copies/reaction and the latter one that can detect copies up to the dilutions of . × copies/reaction. the test was performed using dna from pvp , gpv-chv and several other bacteria and viruses as templates to examine its specificity; the result of this analysis showed that none of the bacteria or viruses (other than gpv-chv and pvp ) yielded any amplification signal, suggesting that the established fq-pcr assay was highly specific (figure ) . the intraassay and interassay cv of this established fq-pcr was in the range of . - % for most of the dynamic range (from . × to . × pvp plasmid copies/ μl). the results demonstrated that the established fq-pcr method was characterized by a wide dynamic range ( logarithmic decades) of detection from . × to . × pvp plasmid copies/μl with high precision. therefore the dynamic range of the method was between . × to . × pvp plasmid copies/μl, which is relatively broad. viral load quantification using the established fq-pcr demonstrated that the gpv dna copy number of each sample could be calculated using the cycle threshold (ct) value determined from the standard curve. the dynamic distribution of gpv within the tissues after oral infection with gpv was intermittently determined by means of the fq-pcr in separate segments of tissues over a -day period. results of this analysis revealed that the blood, heart, liver, spleen, kidney, bursa of fabricius (bf), thymus, and harder's glands were positive at -h postinoculation (pi), with about . - . copies/g. gpv was consistently detected in all the segments of the organs at -h pi. the copy numbers of gpv in each tissue reached a peak at - -h pi. numbers of gpv dna decreased at days, and by days, the level of gpv dna decreased remarkably. importantly, the level of gpv dna was com-parable to that in the other organs at -days pi; the liver, spleen, thymus, harder's glands, and bf had significantly higher numbers of gpv dna than the rest of the tissues, with > copies/g in the former tissues compared to < copies/g in the rest of the tissues. in addition, the control group did not show any positive results at any time point or in any tissue (table ) here, we describe a real-time pcr assay for the quantification of gpv genome coupes in goslings. we confirmed that this assay was highly sensitive, specific, and reproducible. establishment of the fluorescent quantitative real-time pcr (fq-pcr) standard curve real-time pcr has become a potentially powerful alternative in microbiological diagnostics because of its simplicity, rapidity, reproducibility, and high sensitivity compared to other diagnostic methods [ ] [ ] [ ] . in this study, we clearly established the applicability of real-time pcr for the quantification of gpv because of its remarkable sensitivity and high-throughput potential, which is beyond the scope of other diagnostic methods. the real-time pcr assay permits the simultaneous detection and quantification of dna. it is useful for understanding the pathogenesis of the disease and the mechanisms of virus transmission by enabling the investigation of viral dynamics [ ] . the assay can be used to determine the amount of viral dna in different tissues at various times after infection; this infection data could be interesting and useful for expanding the understanding on viruses. quantification of the viral load makes it possible to study the kinetics and tropism of gpv in different birds, tissues, and cells. our study is different from other studies that examined the distribution of viruses and the characteristics of the lesions induced in experimentally infected geese and muscovy ducts by performing comparative pathological studies or other assays [ , ] . previous studies have examined the distribution of gpv in infected muscovy ducks by qualitative pcr [ ] , including a study that used quantitative pcr [ ] . however, bi et al. did not optimize the fq-pcr assay for future application. limn et al. found that gpv could be first detected at -d pi in the liver and other organs. because the real-time pcr method was more sensitive than regular qualitative pcr methods [ ] , we could first detect gpv at -h pi in the liver and other tissues, which was less than h compared to the time required by regular qualitative pcr methods. this finding is important because the prevention and early detection are presently the most logical strategies for virus control [ ] . [ ] . similarly, our study showed that gpv was distributed in the blood, heart, liver, spleen, kidney, bf, thymus, and harder's glands at -h pi. subsequently, gpv was consistently distributed in all the segments of the organs at -h pi. the copy numbers of gpv in the liver, spleen, thymus, harder's glands, and bf was significantly higher than that in the other regions. therefore, these immune organs could be considered as the primary sites of invasion in normal goslings after gpv infection. live gpv vaccine is widely used to immunize adult geese to prevent gpv infection [ ] . real-time pcr and qualitative pcr assays [ ] [ ] [ ] can amplify the highly conserved vp region of the gpv gene, which is distributed in the high-virulence strain and live-vaccine strain of gpv. theoretically, these methods would not be able to differentiate the gpv vaccine strain from the high-virulence strain; nonetheless, we could perform the study on the dynamic distribution of gpv in vivo using these methods, because the animals were certificated as gpv-free by qualitative pcr assay before being infected with the high-virulence strain. for standardization, the vp gene was cloned into a plasmid. the available live vaccine could have been used as the standard. in conclusion, the established real-time pcr assay was rapid, sensitive, and specific for the detection and quantification of gpv dna. in addition, our results provide significant data for clarifying that the immune organs were the primary sites of gpv invasion in infected goslings. template dna was extracted from the viral and bacterial stock solutions using the high pure pcr template preparation kit (roche diagnostics gmbh, mannheim, germany) according to the manufacturer's instructions. the fq-pcr assay primers and probe (namely, gpv-f, gpv-r, and cpv-fp) were designed on the basis of the highly conserved vp region of gpv (genbank accession no. u ). primers and probe were designed by using the primer premier software (version . ). the position and sequence of the primers and probe are shown in table . the product size was bp. the fluorogenic probe was labeled at the ' position with -carboxyfluorescein (fam) dye as a reporter and at the ' position with tetramethylcarboxyrhodamine (tamra) as a quencher and with minor groove binder (mgb™). the sequences of the forward and reverse primers used for the conventional pcr were as described by huang et al., and this primer pair yielded a -bp amplicon [ ] . all the probes and primers were synthesized by takara biotech co., ltd. (dalian, china) and purified by the corresponding high-performance liquid chromatography (hplc) system. the recombinant plasmid dna (namely, pvp ) and primer constructs (namely, vp - and vp - ) were designed to amplify an expected -bp pcr product that included positions , - , bp of gpv (genbank accession no. u ) ( table ). primers were designed by using the primer premier software (version . ). the product was ligated into the pgm-t vector (tiangen corp., beijing, china) and transformed into e. coli dh α competent cells [ ] . the pvp was extracted using the tianprep plasmid extraction kit (tiangen corp., beijing, china). the pvp dna concentration was determined by measuring the absorbance at nm using a smartspec spectrophotometer (bio-rad corp., hercules, ca), and the purity was confirmed using the / nm ratio. on the basis of the molecular weight, we calculated the pvp copy number using the equations described by ke [ ] . the fq-pcr was performed using the abi amplitaq gold dna polymerase system with an icycler iq real-time pcr detection system (bio-rad corp., hercules, ca) according to the manufacturer's instructions. the reaction, data acquisition, and analysis were performed using icycler iq optical system software. the fq-pcr was performed in a -μl reaction mixture containing × pcr buffer, . mmol/l dntps, . u taq, and μl dna template according to the manufacturer's instructions. autoclaved nanopure water was added to make the final volume to μl. each run comprised an initial activation step of s at °c, followed by cycles of denaturation at °c for s and annealing at °c for s; the fluorescence was measured at the end of the annealing/extension step. the tests were performed using . an internal positive control was introduced into the fq-pcr assay to verify that dna was not lost during the extraction step and pcr inhibitors were absent in the dna templates as described by guo et al. [ ] . the fq-pcr standard curve was generated by successive dilutions of pvp with known copy numbers. the purified pvp plasmid dna was serially diluted -fold in te buffer, ph . , from . × to . × plasmid copies/ μl. these dilutions were tested in triplicate and used as quantification standards to construct the standard curve by plotting the plasmid copy number logarithm against the measured ct values. the bio-rad icycler iq detection software was used to generate the standard curve and to calculate the correlation coefficient (r ) of the standard curve and the standard deviations of the triplicate samples. the sensitivities of the conventional pcr and fq-pcr were each determined using triplicates of different concentrations of the recombinant plasmid pvp . template dna was prepared as follows: plasmids of pvp were serially diluted -fold from . × copies/μl to . × copies/μl using sterile ultra pure water. from each dilution, μl was used as a template and subjected to the conventional pcr and fq-pcr protocol. within-run and between-run reproducibilities of the fq-pcr assay were assessed by multiple measurements of pvp samples of different concentrations. the assay was conducted by assessing the agreement between the replicates in five replicates (within-run precision) and in five separate experiments (between-run precision) of the serially diluted pvp plasmid samples through transforming the raw data to their common logarithms and performing analysis of the mean coefficient of variation (cv) values of each pvp standard dilution [ ] . dilutions of pvp plasmid were used to determine the dynamic ranges of the fq-pcr assay. the lower and upper limits of quantification were defined by the pvp recombinant standard plasmid sample concentrations possessing reasonable precision [ ] . gpv-free goslings ( -day-old) that were certificated with qualitative pcr as described by huang [ ] were obtained from the breeding facility of the institute of poultry sciences in sichuan agricultural university, china. animals were bred and maintained in an accredited facility at the institute of poultry sciences in sichuan agricultural university (sichuan, china), and the experiments conducted during this study conform to the principles outlined by the animal welfare act and the national institutes of health guidelines for the care and use of animals in biomedical research. fifty goslings were randomly divided into groups. in brief, a group of goslings were orally infected with gpv ch v strain, using . ml of ld per gosling. another group of goslings was treated with an equal volume of physiologic saline and used as a control [ ] . three goslings from the infected group and gosling from the control group were killed at each time point. blood, heart, liver, spleen, lung, kidney, bf, thymus, esophagus, trachea, brain, harder's glands, duodenum, jejunum, ileum, cecum, and rectum were analyzed by the real-time pcr at different postinoculation (pi) time points, at min; , , , , , and h; and , , , and days. tissues were surgically removed from the goslings and frozen at - °c, weighed, and homogenized using an omni pcr tissue homogenizer (omni). normal tissue sample sizes were mg. for the assays, tissue samples were homogenized in ml of phosphate buffered saline (pbs, ph . ). the homogenizer was washed multiple times between each tissue homogenization. dna was extracted from the tissue samples by using the method described by cheng [ ] . using this assay, we could quantify the viral load. all the samples were analyzed times. the viral concentrations were expressed as the mean log virus genome copy numbers per g or ml of the tested tissue or blood. isolation and identification of goose parvovirus in the uk goose parvovirus-an autonomous member of the dependovirus genus fang dy: recommendation of gpv. veterinary science in china an outbreak of goose parvovirus infection in japan goose parvovirus infection goose parvovirus in england and wales a one-step multiplex real-time rt-pcr for detection and typing of bovine viral diarrhea viruses tissue distribution of the antigenic variants of canine parvovirus type in dogs detection of goose parvovirus genome by polymerase chain reaction: distribution of goose parvovirus in muscovy ducklings distribution of attenuated goose parvoviruses in muscovy goslinglings genetic variation of the nucleocapsid genes of waterfowl parvovirus development and application of pcr to detect goose parvovirus the diagnostic method of pcr for lymphocystis disease(lcd) of cultured paralichthys olivaceus development of a one step real-time rt-pcr method for sensitive detection of human astrovirus development of a real-time reverse transcription polymerase chain reaction assay for detection of marine caliciviruses (genus vesivirus) wutzler p: evaluation of antiviral activity against human herpesvirus (hhv- ) and epstein-barr virus (ebv) by a quantitative real-time pcr assay application of real-time pcr for testing antiviral compounds against lassa virus, sars coronavirus and ebola virus in vitro development of real time pcr for detection and quantitation of dengue viruses application of realtime pcr to quantify hepatitis b virus dna in chronic carriers in the gambia detection of goose parvovirus distribution in geese by fluorescence quantitative pcr assay real-time pcr in virology study on the gastrointestinal tract distribution of salmonella enteritidis in orally infected mice with a species specific fluorescent quantitative pcr realtime pcr assay in leishmania-infected dogs treated with meglumine antimoniate and allopurinol fluorescent and electronmicroscopy immunoassays employing polyclonal and monoclonal antibodies for detection of goose parvovirus infection comparative pathological studies on domestic geese (anser anser domestica) and muscovy ducks (cairina moschata) experimentally infected with parvovirus strains of goose and muscovy duck origin new real-time pcr tests for species-specific detection of chlamydophila psittaci and chlamydophila abortus from tissue samples development of taqman mgb fluorescent real-time pcr assay for the detection of anatid herpesvirus an immunocytochemical study on the sequential tissue distribution of duck plague virus development of a quantitative light cycler real-time rt-pcr for detection of avian reovirus development and application of a reverse transcriptasepolymerase chain reaction detect chinese isolates of duck hepatitisvirus type the authors declare that they have no competing interests. jy carried out most of the experiments and wrote the manuscript. ac and mw critically revised the manuscript and the experiment design. kp, ml, yg, cl, dz and xc helped with the experiment. all of the authors read and approved the final version of the manuscript. key: cord- -dt jku v authors: hon, kam le; ng, pak c; leung, ting f title: influenza or not influenza: analysis of a case of high fever that happened years ago in biblical time date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: dt jku v the bible describes the case of a woman with high fever cured by our lord jesus christ. based on the information provided by the gospels of mark, matthew and luke, the diagnosis and the possible etiology of the febrile illness is discussed. infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. if the postulation is indeed correct, the woman with fever in the bible is among one of the very early description of human influenza disease. infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. we analysed a case of high fever that happened years ago in biblical time and discussed possible etiologies. the bible descrbies the case of a woman with high fever cured by our lord jesus christ. according to mark : to and matthew : - , the mother-in-law of simon peter "lay sick" with a febrile illness [ ] . when jesus took her by the hand and lifted her up, the fever immediately left. the lady began to serve the household and probably prepared a meal. the case is also described in the gospel by luke (luke : - ), who was a physician in his days and he specifically mentioned that the fever was high [ ] . what was the diagnosis of the febrile illness, based on the information provided by the gospels of mark, matthew and luke [ ] ? it seems that the woman suffered an acute febrile illness with high fever and was sick enough to be bed-ridden. luke did not quantify the fever as the fahrenheit temperature scale was not invented until [ ] . no other symptom or chronic illness was described in the three gospels. possible etiology of her "acute febrile illness" is some sort of infection or inflammation. the bible describes that when jesus touched the woman, the fever retreated instantaneously. this implies that the disease was probably not a severe acute bacterial infection (such as septicemia) or subacute endocarditis that would not resolved instantaneously. it was probably not an autoimmune disease such as systemic lupus erythematousus with multiple organ system involvement, as the bible does not mention any skin rash or other organ system involvement. the instantaneous cure also makes an underlying malignant etiology unlikely. it seems that an acute selflimiting infectious illness is a possible diagnosis. the brief duration, high fever, and abrupt cessation of fever makes influenza disease probable [ ] . shortly following her recovery, presumbly within minutes, it is described that the woman began to serve jesus and the disciples, thus making influenza illness highly probable. most miserably sick patients recover without sequlae when the high fevers subside following influenza-like illness [ ] . the next question is whether the virus is influenza, avian flu, parainfluenza, or other respiratory viruses such as adenovirus or even sars-cov (severe acute respiratory syndrome-associated coronavirus) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . adenovirus and sars-cov are usually associated with pulmonitis, and the pulmonary symptoms may not resolve promptly [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is unable to tell if the woman has been in contact with poultry or swine and contracted avian or swine influenza [ , ] . the bible does not describe if any members of the family including andrew and simon developed febrile illness, before or subsequent to her febrile illness. the characteristic features of seasonal influenza include abrupt onset of fever, chills, non-productive cough, myalgias, headache, nasal congestion, sore throat, and fatigue. the diagnosis is mainly clinical. seasonal influenza would be less likely if no members of the family were affected [ ] . avian influenza and other respiratory viruses may cause isolated infection without efficient human-to-human transmission. in any case, influenza-like illness due to a respiratroy virus would explain her symptomatology and clincial course [ ] . other possibilities include drug fever and poisoning (such as atropine). naturally-occurring plants containing the belladonna alkaloid atropine could have been consumed but the bible does not describe unusual food or medicine intake by the woman and her family. the other side effects of anticholinergic agent were absent. the woman would recover spontaneously when the effect of the offending substance wore off. one final consideration that one might have is whether the illness was inflicted by a demon or devil. the bible always tells if an illness is caused by a demon or devil (matthew : - , : , : - ; mark : - , : - , : - ; luke : - , : - , : - , : ) [ ]. the victims often had what sounded like a convulsion when the demon was cast out. in our index case, demonic influence is not stated, and the woman had no apparent convulsion or residual symptomatology. the bible has many examples of descriptions of medical diseases. for instance, the first pediatric case of mouth-to-mouth cardiopulmonary resuscitation is vividly described in the old testament when the prophet elisha pressed upon an apparently dead child and breathed into him seven times, and the child was revived (kings : - ) [ ] . influenza and respiratory viral infections have been documented throughout human history [ ] . the current flu pandemic is a global outbreak of a new strain of h n influenza virus, often referred to colloquially as "swine flu" which began in the state of veracruz, mexico in april and the virus continued to spread globally. the world health organization (who) and us centers for disease control (cdc) in june escalated the global alert level to phase and declared the outbreak to be a global pandemic since the hong kong flu [ ] . if the postulation is indeed correct, the woman with fever in the bible is among one of the very early description of human influenza disease. new king james version fahrenheit temperature scale. sizes, inc severe childhood respiratory viral infections just like sars clinical presentations and outcome of severe acute respiratory syndrome in children personal view of sars: confusing definition, confusing diagnoses severe acute respiratory syndrome (sars) in children: epidemiology, presentation and management severe acute respiratory syndrome: 'sars' or 'not sars premorbid factors and outcome associated with respiratory virus infections in a pediatric intensive care unit influenza or not influenza: analysis of a case of high fever that happened years ago in biblical time authors' contributions klh conceived of the study, and was the principal author. tfl and pcn advised and reviewed the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -ekxsv t authors: yu, yunjia; zhang, yang; wang, shuyao; liu, wei; hao, cui; wang, wei title: inhibition effects of patchouli alcohol against influenza a virus through targeting cellular pi k/akt and erk/mapk signaling pathways date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: ekxsv t background: patchouli alcohol (pa) is a tricyclic sesquiterpene extracted from pogostemonis herba, which is a traditional chinese medicine used for therapy of inflammatory diseases. recent studies have shown that pa has various pharmacological activities, including anti-bacterial and anti-viral effects. methods: in this study, the anti-influenza virus (iav) activities and mechanisms were investigated both in vitro and in vivo. the inhibitory effects of pa against iav in vitro were evaluated by plaque assay and immunofluorescence assay. the neuraminidase inhibition assay, hemagglutination inhibition (hi) assay, and western blot assay were used to explore the anti-viral mechanisms. the anti-iav activities in vivo were determined by mice pneumonia model and he staining. results: the results showed that pa significantly inhibited different iav strains multiplication in vitro, and may block iav infection through inactivating virus particles directly and interfering with some early stages after virus adsorption. cellular pi k/akt and erk/mapk signaling pathways may be involved in the anti-iav actions of pa. intranasal administration of pa markedly improved mice survival and attenuated pneumonia symptoms in iav infected mice, comparable to the effects of oseltamivir. conclusions: therefore, patchouli alcohol has the potential to be developed into a novel anti-iav agent in the future. influenza a virus (iav) belongs to the orthomyxoviridae family, being segmented, single stranded, negative sense rna viruses [ ] . iav caused at least three large-scale influenza outbreaks in the twentieth century, the most serious of which was the spanish influenza outbreak in , which caused more than million deaths [ , ] . more recently, the emergence and global spread of h n influenza from the pandemic and recent lethal cases of h n and h n influenza demonstrate the limitations of currently available strategies to control influenza infection [ ] . currently, there are three main types of anti-iav drugs approved for clinical use: ( ) m ion channel inhibitors such as amantadine and rimantadine; ( ) neuraminidase inhibitors such as oseltamivir, zanamivir, and peramivir [ , ] ; ( ) polymerase inhibitors such as baloxavir. however, the emergence of drugresistant influenza variants such as amantadine and oseltamivir resistant iav strains has led to a decline in the efficacy of these drugs. in addition, most of these anti-iav drugs also have some side effects such as nervous system damage [ ] [ ] [ ] . therefore, new influenza therapeutics with novel mechanisms of action are urgently required to combat the persistent threat of influenza viruses. patchouli alcohol is a tricyclic sesquiterpene extracted from pogostemonis herba, which has long been used in the treatment of inflammatory diseases as a traditional chinese medicine [ ] . recent studies have shown that patchouli oil has various pharmacological activities, including anti-emetic [ ] , anti-inflammatory [ ] , antibacterial [ ] , and anti-viral effects [ ] . li et al. found that oral administration of pa appeared to be able to augment protection against influenza virus infection in mice via enhancement of host immune responses, and attenuation of systemic and pulmonary inflammatory responses [ ] . wu and co-workers reported that patchouli alcohol inhibited influenza a (h n ) virus mainly through interfering with the functions of virus neuraminidase [ ] . therefore, pa has the potential to be developed into a novel anti-viral agent in the future. to further correlate the potential anti-iav applications of pa with its underlying molecular mechanisms, the anti-iav (h n ) effects and mechanisms of pa were investigated in vitro and in vivo in this study. the results showed that pa may block iav infection through inactivating iav directly and interfering with some early steps after virus adsorption. cellular pi k/akt and erk/ mapk signaling pathways may be involved in the anti-iav actions of pa. in addition, intranasal administration of pa markedly improved mice survival and attenuated pneumonia symptoms in iav infected mice. patchouli alcohol (pa) (with purity > %) was purchased from targetmol (shanghai, china). dulbecco's modified eagle's medium (dmem), penicillin, and streptomycin were purchased from gibco (grand island, ny, usa). fetal bovine serum (fbs) was obtained from excell (suzhou, china). mouse anti-influenza a virus np antibody and alkaline phosphatase (ap)-labeled secondary antibodies were obtained from santa cruz biotechnology (usa). dylight conjugated secondary antibody was obtained from abbkine (california, usa). ribavirin injection ( mg/ml) was purchased from lukang cisen (jining, china). oseltamivir carboxylate was purchased from santa cruz biotechnology (santa cruz, ca, usa). oseltamivir phosphate was obtained from roche (shanghai, china). the influenza neuraminidase inhibitor detection kit was purchased from beyotime (shanghai, china). the anti-np protein was provided by abcam (ab ), and other antibody, such as anti-phosphorylated pi k( s), akt ( s), mtor ( s), erk / ( s), and nf-κb ( s) antibodies, or anti-gapdh ( s) and α-tubulin antibodies ( s), were obtained from cell signaling technology (danvers, usa). madin-darby canine kidney (mdck) cells were grown in dmem medium supplemented with % fbs, u/ml of penicillin and μg/ml of streptomycin. a cells were cultivated in f medium containing % fbs and mm l-glutamine. influenza a virus h n (a/puerto rico/ / ), h n (a/nws/ ), and h n (a/virginia/ atcc / ) were propagated in -day-old embryonated eggs for days at . °c. for infection, virus propagation solution was diluted in pbs containing . % bovine serum albumin (bsa) and was added to cells at the indicated multiplicity of infection (moi). virus was allowed to adsorb min at °c. after removing the virus inoculum, cells were maintained in infecting media (dmem, μg/ml trypsin) at °c in % co . the cytotoxicity of compounds was measured by the mtt (sigma-aldrich, usa) assay. confluent mdck, a and ft cell cultures in -well plates were exposed to different concentrations of pa ( . , . , , , μg/ml) in triplicate for h. after that, ul of pbs containing mtt (final concentration: . mg/ml) was added to each well. after h incubation at °c, the supernatant was removed and ul of dmso was added to each well to solubilize the formazan crystals. after vigorous shaking, absorbance values were measured in a microplate reader (bio-rad, usa) at nm. the cc was calculated as the compound concentration necessary to reduce cell viability by %. different concentrations ( , , . , . , . or . μg/ml) of pa in μl dmem media were mixed with an equal volume of infectious iav ( - pfu/ well) in dmem, and incubated at °c for h. the virus-pa mixtures were then transferred to confluent mdck cell monolayers in -well plates, and incubated at °c for h with gentle shaking every min. after that, the inoculum was removed and each well was overlaid with ml of agar overlay medium ( . % agarose, . % deae dextran, mm l-glutamine, . mm nonessential amino acids, u/ml penicillin, mg/ml streptomycin and mg/ml tpck treated trypsin). after incubation for days at °c in % co , cells were fixed with % para-formaldehyde (pfa), followed by staining with % crystal violet for plaque counting. mdck cells were infected with h n (vir , moi = . ) under four different treatment conditions. i) pretreatment of virus: iav was pretreated with μg/ml of pa at °c for h before infection. ii) pretreatment of cells: mdck cells were pretreated with μg/ml of pa at °c for h before infection. iii) adsorption: mdck cells were infected in media containing μg/ml of pa and, after h adsorption at °c, were overlaid with compound-free media. iv) after adsorption: after h adsorption at °c, the inoculum was removed and the infecting media containing μg/ml of pa were added to cells. at h p.i., the antiviral activity was determined by plaque assay. mean percentage virus titers were calculated as a percentage of plaque titers from untreated control group. iav virus (moi = . ) infected a cells were treated with or without pa ( , μg/ml) after adsorption. at h post infection, mdck cells were fixed with % pfa for min. then cells were permeabilized and incubated sequentially with primary antibodies against iav np protein and dylight conjugated secondary antibody. then after washing, the cell nucleus was stained with dapi for min before confocal imaging. images were recorded using a nikon confocal microscope, and analyzed by imagej (nih) version . u (usa). the hemagglutination (ha) assay was performed as previously reported [ ] . standardized chicken red blood cell (crbc) solutions were prepared according to the who manual. virus propagation solutions were serially diluted -fold in round bottomed -well plate and % crbcs were then added at an equal volume. after min incubation at °c, rbcs in negative wells sedimented and formed red buttons, whereas positive wells had an opaque appearance with no sedimentation. ft cells were cultured in well plates at °c for h before transfected with plasmids (pb /pcdna . , pb /pcdna . , pa/pcdna . , or np/pcdna . ) encoding pr virus polymerase subunits (pa, pb , and pb protein) and virus nucleoprotein (np), and a luciferase rna expression vector (vns -luc/phh ). then the cells were treated with or without pa ( . , . , . , and μg/ml) or nucleozin ( μm). the effect of vrna transcription was then evaluated by measuring luciferase activity according to manufacturer's instructions after incubation at °c for h. the influenza neuraminidase inhibitor detection kit was used to measure the inhibition of na activity [ ] . briefly, inactivated pr virus supernatants was added to a -well plate and then mixed with different compounds (diluted in mm mes buffer (ph . ), mm cacl ) at °c for min. then munana ( μm) was added as the substrate and incubated at °c for min. the reaction was stopped by the addition of stop solution ( % ethanol, . m glycine, ph . ). fluorescence was measured using a spectramax m plate reader with excitation and emission wavelengths of and nm, respectively. total rna was extracted from vir virus (moi = . ) infected mdck cells using an rnaiso™ plus kit (takara, japan), and analysed by using the one step sybr prime-script rt-pcr kit (takara, japan). the real-time rt-pcr was performed using the following primers: virus ha mrna, ′-aagtcctcgtgctatggg- ′ and ′-tgggaggctggtgtttat- ′; β-actin mrna, ′-ctccatcctggcctcgctgt- ′ and ′-gctgtc accttcaccgttcc- ′. the real-time rt-pcr was performed at °c min, °c s, cycles of °c s, °c s, followed by melting curve analysis, according to the instrument documentation (abi prism , applied biosystems, usa). the relative amounts of virus ha mrna molecules were determined using the comparative ( -ΔΔct ) method, as previously described ( ) . after drug treatment, the cell lysate was separated by sds-page and transferred to nitrocellulose membrane. after being blocked in tris-buffered saline (tbs) containing . % tween (v/v) and % bsa (w/v) at room temperature for h, the membranes were rinsed and incubated at °c overnight with anti-np protein (santa cruz, usa), anti-phosphorylated pi k, akt, erk / , and nf-κb antibodies, or anti-gapdh, β-actin, and αtubulin antibodies (cell signaling technology, danvers, usa) as control. the membranes were washed and incubated with ap-labeled secondary antibody ( : dilutions) at rt for h. the protein bands were then visualized by incubating with the developing solution (pnitro blue tetrazolium chloride (nbt) and -bromo- chloro- -indolyl phosphate toluidine (bcip) at rt for min. the relative densities of proteins were all determined by using imagej (nih) v. . u (usa). four-week-old female kunming mice (average weight, . ± . g) were housed and studied under protocols approved by the animal care and use committee of ocean university of china (oucyy- ). mice received humane care in accordance with the guidelines provided by the national institutes of health for the use of animals in laboratory experiments. mice per group were inoculated intranasally with pr ( ld /mouse) diluted in ul of × pbs. all the mice were randomly divided into experimental groups. four hours after inoculation, mice received intranasal therapy of pa ( or μg/day) or oral therapy of oseltamivir phosphate ( mg/kg/day), and the treatments were repeated once daily for days. mice were weighed and killed on day after inoculation, and the lungs were then removed, weighed, and homogenized in × pbs for determination of viral titers by plaque assay. histopathological analysis was performed using h&e staining on samples collected on days post infection (dpi) as described previously [ ] . in the survival experiments, mice per group were intranasally infected with pr/ virus ( ld /mouse) at day . the drugs administration was repeated once daily during the experiment, and survival was assessed in all groups for days after infection. mice were monitored daily for weight loss and clinical signs. if a mouse lost body weight over % of its pre-infection weight, it was defined as dead and humanely euthanized immediately; the rest of the mice were sacrificed at the end of experiment on dpi. all data are representative of at least three independent experiments. data are presented as means ± standard deviations (sd). statistical significance was calculated by graphpad prism software using one-way anova with turkey's test, with p values < . considered significant. the cytotoxicity of patchouli alcohol (pa) was firstly evaluated by mtt assay in mdck, a and ft cells [ ] . the results showed that pa exhibited no significant cytotoxicity at the concentrations from . to μg/ml (fig. a) . the cc ( % cytotoxicity concentration) values for pa in mdck, ft and a cells were about . , . , and . μg/ml, respectively. these results were used to determine the dose range of pa for the subsequent experiments. pa was then assayed for its ability to inhibit iav multiplication in vitro using plaque assay [ ] . firstly, the inhibition of pa on the virus yields from mdck cells infected with vir (a/virginia/atcc / ), nws (a/nws/ ) or pr (a/puerto rico/ / ) at high moi (≈ . pfu/cell) were examined by plaque assay. as shown in fig. b and c, pa treatment reduced the virus titers of vir , nws, and pr in a dose-dependent manner when used at the concentrations of . - μg/ ml. the % inhibitory concentration (ic value) of pa for vir , nws, and pr was about . ± . , . ± . , and . ± . μg/ml, respectively (table ) . at the concentration of . μg/ml, the virus titers reduced about fold of that in the untreated control group for vir , . fold of that for nws, and . fold of that for pr virus (fig. b and c) . to further explore whether pa had direct inhibition actions on viral particles, the plaque reduction assay was performed as previously described [ ] . in brief, vir virus ( - pfu/well) was pre-incubated with or without pa for min at °c before infection. ten the virus-pa mixture was transferred to confluent cell monolayers in -well plates incubated at °c for h and subjected to plaque assay. as shown in fig. d and e, pre-incubation of pr with pa at the concentrations of . and μg/ml markedly reduced the number of plaques and protected mdck cells, suggesting that pa may be able to inactivate viral particles directly. furthermore, the inhibition effects of pa on iav infection was also examined over multiple cycles of infection using plaque reduction assay [ ] . briefly, mdck cells were infected with pa pretreated virus (vir , nws, and pr ) at an moi of . pfu for h at °c, and then subjected to plaque assay. as shown in fig. f , pa also significantly inhibited the plaque formation in vir , nws and pr (moi = . ) infected cells when used at the concentration > . μg/ml (fig. f) . the ic values of pa for vir , nws, and pr was about . ± . , . ± . , and . ± . μg/ml, respectively (table ) . however, ribavirin could not significantly inhibit the plaque formation of vir with ic value > μg/ml (table ) . thus, pa possessed anti-iav effects in vitro, and the pandemic h n virus (vir ) was most susceptible to pa treatment. various time-points were assessed to determine the stage(s) at which pa exerted its inhibitory effects in vitro. briefly, mdck cells were infected with vir virus (h n ) (moi = . ) under four different treatment conditions: pre-treatment of viruses, pre-treatment of cells, during adsorption, or after adsorption. at h p.i., the antiviral activity was determined by plaque assay. as shown in fig. a , pretreatment of vir virus with μg/ml pa for h before infection markedly reduced virus titers, suggesting that pa may have direct interaction with iav particles. however, either the addition of pa during adsorption or pretreatment of cells only weakly inhibited virus multiplication (fig. a) , suggesting that pa may not interact with mdck cells directly. interestingly, treatment of pa after adsorption also significantly reduced virus titers as compared to the nontreated virus control group (fig. a) . thus, pa may be able to inactivate virus particles directly and block some stages after virus adsorption. moreover, another time course study was also performed to explore which viral stage after adsorption is inhibited by pa as described previously [ ] . briefly, (fig. b ). since pa may be able to inactivate virus particles directly, we then explored whether pa had direct interaction with virus surface na and ha protein by using the neuraminidase inhibition assay and hemagglutination inhibition (hi) assay. as shown in fig. c , pa could not significantly inhibit the na activities of vir virus at the concentrations of . - μg/ml, while zanamivir possessed high inhibition percentage (> %) at μg/ ml, suggesting that pa may have no direct interaction with virus na protein. moreover, the results of the hi assay showed that the anti-ha antibodies significantly inhibited the pr virus-induced aggregation of chicken erythrocytes at the concentrations of . - μg/ml (fig. d) , suggesting that the anti-ha antibody can block the virus attachment to red blood cells through binding to ha. however, pa did not obviously inhibit virusinduced aggregation of chicken erythrocytes even at a concentration of μg/ml (fig. d) , suggesting that pa may have no direct interaction with viral ha protein. furthermore, we also performed mini-genome assay to evaluate the influence of pa on viral genome replication, which occur during the early stages in viral life cycle. briefly, ft cells were transfected with four expression plasmids encoding pr virus pb , pb , pa, and np proteins, and the luciferase-containing plasmid vns luc/phh , which encodes a viral-like genome in the absence or presence of pa ( . , . , . , and μg/ml). the effect of vrna transcription was then evaluated by measuring luciferase activity at h p.i. the results showed that the positive drug nucleozin caused a notable reduction in luciferase activity at μm as compared with control (dmso) treatment (fig. e) . by contrast, treatment with pa ( . , . , . , and μg/ml) did not significantly inhibit luciferase activity, suggesting that np protein may be not the direct target of pa. in summary, virus ha, na and np proteins may not be the main targets of pa in vitro. since pa may inhibit some steps after virus adsorption ( fig. a and b) , the effects of pa on viral protein synthesis and rna replication were evaluated by using immunofluorescence assay and real-time rt-pcr assay as described previously [ ] . firstly, vir virus (moi = . ) infected a cells were added with or μg/ml of pa after virus adsorption and then incubated at °c for h. after that, viral np protein expression was detected by immunofluorescence assay. as shown in fig. a , in virus-infected cells without drug treatment, the fluorescence of viral np proteins could be obviously found in both the cell nucleus and cytoplasm (fig. a) , while nearly non fluorescence could be found in the noninfected cells (fig. a) . however, after treatment with pa for h, the number of virus antigen-expressing cells was drastically reduced, and only very few fluorescence could be found in the cytoplasm (fig. a) . quantitation of data of the fluorescence intensity in iav infected cells showed that pa treatment ( , μg/ml) significantly reduced the fluorescence intensity of np in a cells, suggesting that pa may block some steps of iav life cycle after adsorption to interfering with nuclear import and expression of np protein (fig. b) . moreover, the inhibition effect of pa on virus mrna expression was then evaluated by real-time rt-pcr assay. iav (moi = . ) infected cells were added with pa ( , , μg/ml) after virus adsorption and then incubated at °c for h. after that, total rna was extracted for real-time rt-pcr. as shown in fig. c , after treatment with pa ( , μg/ml) for h, the iav np mrna levels decreased to about . and . % of that of untreated cells after pa treatment, respectively, consistent to the results of immunofluorescence assay. furthermore, western blot assay was also performed to verify the inhibition of pa on viral protein production. mdck cells were firstly infected with iav (moi = . ), and then treated with or without pa at indicated concentrations after adsorption. after incubation for h, viral np protein production was detected by western blot assay. as shown in fig. d and e, the level of viral np protein was significantly reduced by pa in a dosedependent manner as compared to that of the nontreated virus control group (pr ) (p < . ). the treatment with pa at μg/ml reduced the production of iav np protein by more than % (fig. e ). therefore, pa may also be able to inhibit iav protein and mrna expression through interfering with some early steps of virus life cycle. since pa may inhibit some steps after virus adsorption to reduce iav mrna and protein expression in vitro, so we further explored if pa could influence some cellular signaling pathways required for iav infection. the cellular pi k/akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of pi k/akt signaling could inhibit both entry and replication of virus [ , ] . in this study, after iav infection for h, the levels of phosphorylated pi k proteins were significantly increased to about . fold higher than normal control group in iav infected cells (p < . ) (fig. a and e) . however, after treatment with pa ( . , . , , μg/ml) for h, the expression level of phosphorylated pi k significantly decreased from about . to about . , . , . , and . -fold of normal control group, respectively (p < . ) (fig. a and e) . moreover, the activation of pi k can induce the activation of some downstream signals such as akt, and the level of phosphorylated akt was truly significantly increased in virus-control group to about . fold higher than normal control group at h p.i. (p < . ) (fig. b and f ). but treatment with pa ( , μg/ml) for h could significantly reduce the activation of akt from about . to about . and . -fold of normal control group, respectively ( fig. b and f) . thus, the pi k/akt signaling pathway may be involved in the anti-iav mechanisms of pa in vitro. furthermore, the mapk signaling pathway was reported to be required for efficient vrnp export from nucleus, and the inhibitors of mapk pathway could reduce iav replication and inflammatory symptoms [ ] [ ] [ ] . in this study, erk / protein was significantly activated in virus-control group to approximately . fold higher than normal control group at h p.i. (p < . ) ( fig. c and g). however, after treatment with pa ( . , . , and μg/ml) for h, the expression level of phosphorylated erk / protein significantly decreased from about . to about . , . , . , and . -fold of normal control group, respectively (p < . ) ( fig. c and g) . however, treatment with pa ( . , . , and μg/ml) for h could not significantly reduce the expression level of phosphorylated nf-κb protein as compared to the virus control group (fig. d and h) . thus, pa may inhibit erk/mapk rather than nf-κb pathway to interfere with iav replication. moreover, the pi k/akt pathway was reported to be associated with host antiviral response [ , ] , so we further explored the influence of pa on immune response by using western blot and elisa assay. we first evaluate the direct actions of pa on cellular pi k/akt pathway in non-infected a cells using western blotting. the results showed that pa treatment ( . , . , , μg/ml) could not significantly influence the activation of pi k and akt proteins in the non-infected a cells ( fig. a and b) , suggesting that the inhibition of pi k/akt pathway by pa may be related to its inhibition of iav infection. treatment of pa for different time intervals within h showed no significant cytotoxicity to noninfected a cells (fig. c ). in addition, iav infection significantly increased the production of cellular interferon-β (ifn-β) in vir virus infected a cells, however, pa treatment ( . , . , , μg/ml) could not significantly influence the production of ifn-β as compared to the virus control group (fig. d) , suggesting that pa had no direct action on cellular antiviral response. furthermore, we also evaluated the influence of pa on the production of interferon-γ (ifn-γ) and interleukin (il- ) in mice with or without pr virus infection. as shown in fig. e , intranasal treatment of pa ( or μg/day) for four days had no significant influence on the production of ifn-γ and il- in non-infected mice. however, pa treatment could significantly reverse the reduction of ifn-γ and il- in iav infected mice (fig. f) , suggesting that the enhancement of pa on type-ii interferon system may be related to its inhibition of iav inhibition in vivo. thus, the inhibition of pi k/akt pathway by pa may be related to its inhibition of iav infection rather than direct actions on host antiviral response. the anti-iav effects of pa in vivo were further explored using a mouse pneumonia model [ ] . in brief, iav-infected mice received intranasal administration of pa ( or μg/day) or placebo (pbs) once daily for the entire experiment, and the selected subset of treated, infected mice were then sacrificed on day and the tissue samples were removed for further analysis. subsequently, the pulmonary viral titers were determined by plaque assay [ ] . as shown in fig. a , after treatment of pa ( , μg/day) for days, the pulmonary viral titers significantly decreased compared to that of the virus control group (p < . ), suggesting that intranasal therapy with pa could inhibit iav multiplication in mice lungs. oral therapy of oseltamivir ( mg/kg/day) also showed significant reduction of virus titers in mice lungs (p < . ) (fig. a) . moreover, the survival experiments were also performed to evaluate the effects of pa on the survival of iav-infected mice. as shown in fig. b , intranasal administration with pa ( μg/day) significantly increased survival rates as compared to the placebo-treated control group (p < . ). by day post infection, only % of the individuals in the placebo group survived whereas % of animals in the pa ( μg/day)-treated group survived, superior to that in oseltamivir ( mg/kg/day)treated group ( %). pa treatment at μg/day also increased the survival rate of iav infected mice ( %) but without significance (fig. b) . to further evaluate the effects of pa on viral pneumonia in mice, histopathology analysis was also performed (see figure on previous page.) fig. the influence of patchouli alcohol on virus protein and mrna expression. a immunofluorescence assay of virus np protein in h n (vir ) infected a cells at h p.i. scale bar represents μm. b the average fluorescence intensity of np proteins in (a) was measured by imagej (nih) version . u (usa) to calculate the average intensity per unit area of cells of different images (n = ). significance: * p < . , * * p < . vs virus control group. c vir (moi = . ) infected mdck cells were treated with different concentrations of pa ( - μg/ml), and incubated at °c for h. after that, total rna was extracted for real-time rt-pcr assay of iav ha mrna and cellular β-actin mrna. the relative amounts of virus ha mrna were determined using the comparative ( -ΔΔct ) method. rna levels for non-drug treated cells (virus control) were assigned values of . values are means ± sd (n = ). significance: *p < . vs. virus control group. d mdck cells were firstly infected with iav (moi = . ), and then treated with or without pa at indicated concentrations after adsorption. at h p.i., the virus np protein expression was evaluated by western blotting. blots were also probed for β-actin protein as loading controls. e quantification of immunoblot for the ratio of iav np protein to actin. the ratio for non-treated virus control group (pr ) were assigned values of and the data presented as mean ± sd (n = ). significance: **p < . vs. virus control group (pr ) as described previously [ ] . as shown in fig. c , lung tissues in virus-control group showed marked infiltration of inflammatory cells in the alveolar walls and the presence of massive serocellular exudates in the lumen. however, after treatment with pa ( or μg/day) for days, the lung tissues showed intact columnar epithelium in the bronchiole even in the presence of some serocellular exudates in the lumen (fig. c ). mice treated with oseltamivir ( mg/kg/day) also had intact columnar epithelium (fig. c) . thus, pa may be able to attenuate pneumonia symptoms in iav infected mice. natural products from chinese medicine have been attracting more and more attention of pharmacists. patchouli alcohol (pa), a tricyclic sesquiterpene extracted from pogostemonis herba, was reported to possess anti-viral activities against different viruses especially influenza virus [ ] [ ] [ ] . in the current study, we found that pa could inhibit different influenza a virus replication in vitro, and the pandemic h n virus (vir ) was most susceptible to pa treatment (ic < . μg/ml). intranasal administration of pa significantly promoted the survival rate of mice and attenuated pneumonia symptoms in iav infected mice, comparable to the positive control drug oseltamivir. thus, patchouli alcohol merits further investigation as a novel anti-iav agent in the future. the time-of-addition assay indicated that pretreatment of iav with pa before infection or addition of pa during adsorption markedly reduced virus multiplication (fig. ) , suggesting that pa may have direct inactivation effects on iav particles. pa was reported to inhibit h n virus replication mainly through inhibition of the functions of virus neuraminidase [ ] . however, in contrast to the previous studies, we found that pa could not significantly inhibit the na activity of h n virus, and did not significantly block ha mediated aggregation of chicken red blood cells. thus, pa may be not able to directly bind to virus surface proteins but may interfere with the interaction between iav and cell receptors. interestingly, post-treatment of cells with pa after adsorption also dramatically inhibited virus multiplication, suggesting that pa may also block some stages after virus adsorption. cellular pi k/akt signaling pathway is known to be able to augment replication of several viruses, and may be associated with lytic infections of both rna and dna viruses, including influenza a virus [ ] . some inhibitors of pi k or its downstream signal akt could significantly block virus entry and replication [ ] [ ] [ ] . herein, patchouli alcohol was found to be able to significantly inhibit the phosphorylation of pi k and akt proteins in iav-infected cells (fig. ) , suggesting that pa may inhibit the activation of pi k/akt signaling pathway to block virus infection and replication. however, pa could not influence the activation of pi k/akt pathway in non-infected a cells and could not directly enhance the interferon system in vitro, suggesting that the inhibition of pi k/akt pathway by pa may be related to its inhibition of iav infection rather than direct actions on host antiviral response. moreover, the mapk and nf-κb signaling pathways were reported to be required for efficient vrnp export from nucleus and virus rna synthesis, and the inhibitors of mapk pathway could reduce both iav replication and inflammatory symptoms [ ] [ ] [ ] . in this study, pa significantly reduced the activation of erk / rather than nf-κb in iav infected cells, suggesting that erk/mapk rather than nf-κb pathway may be involved in the anti-iav actions of pa. considered that pa could significantly inhibit virus mrna and protein expression in iav infected cells, we posit that pa may interfere with the activation of pi k/akt and erk/mapk signaling pathways, thus inhibiting the invasion and subsequent replication of iav. the in vivo anti-iav effects of pa were also explored in a murine pneumonia model of influenza. intranasal treatment of pr -infected mice with pa markedly improved their survival and decreased the pulmonary virus titers (fig. ) . moreover, the histopathological analysis indicated that pa treatment also (see figure on previous page.) fig. the influence of patchouli alcohol on host antiviral response. a a cells were treated with or without pa ( . , . , , μg/ml) for h, and then the phosphorylation of pi k and akt proteins was evaluated via western blotting. blots were also probed for β-actin and gapdh protein as loading controls. the result shown is a representative of three separate experiments. b plots quantifying the immunoblots (as ratios to β-actin or gapdh) for p-pi k and p-akt proteins, respectively. the ratios for non-treated cells (mock) were assigned values of . and the data presented as mean ± s.d. (n = ). c a cells were treated with patchouli alcohol ( , μg/ml) for specified time period, and then the media were removed and cells were overlaid with compound-free media. then at h p.i., the cell viability of a cells was measured by mtt assay. values are means ± s.d. (n = ). d vir virus (moi = . ) infected cells were treated with or without pa ( . , . , , μg/ml) for h, then the content of ifn-β in the culture supernatants was detected using elisa kits. values are means ± s.d. (n = ). ##p < . vs. non-infected group (mock control). e and f after treatment of pa ( or μg/day) for four days in non-infected mice (e) or pr virus infected mice (f), the production of interferon-γ (ifn-γ) and interleukin (il- ) in lung tissues was determined by using the elisa kits for ifn-γ and il- . values are means ± s.d. (n = ). significance: ##p < . vs. non-infected mock control group; **p < . vs. virus control group attenuated the pneumonia symptoms in iav-infected lungs, comparable to the effects of oseltamivir. however, pa treatment exerted obvious therapeutic effect only when starting earlier ( h p.i.), which will restrict its clinical application to some extent. different to the oral administration of oseltamivir, pa was administrated through intranasal, and low dose therapy of pa ( μg/day) had comparable anti-iav effects to fig. the anti-iav effects of patchouli alcohol in vivo. a viral titers in lungs. after treatment with oseltamivir ( mg/kg/day) or pa ( or μg/ day) for days, the pulmonary viral titers were evaluated by plaque assay. values are the mean ± sd (n = ). significance: *p < . , **p < . vs virus control group. b survival rate. iav infected mice received therapy with oseltamivir ( mg/kg/day) or pa ( or μg/day) for the entire experiment. results are expressed as percentage of survival, evaluated daily for days. significance: *p < . vs. virus control group (placebo). c histopathologic analyses of lung tissues on day p.i. by he staining (× ). the representative micrographs from each group were shown (n = mice/group). mock: non-infected lungs; control: iav infected lungs without drugs; oseltamivir: iav infected lungs with oseltamivir ( mg/kg/day) treatment; pa μg/day: iav infected lungs with pa ( μg/day) treatment; pa μg/day: iav infected lungs with pa ( μg/day) treatment. the red arrows indicate the presence of inflammatory cells in the alveolar walls and serocellular exudates in the lumen oseltamivir ( mg/kg/day), suggesting that pa may be used alone or combined with oseltamivir for treatment of influenza by different administration. in summary, pa possesses anti-iav activities both in vitro and in vivo, and may block iav infection through targeting virus particles and cellular pi k/akt and erk/mapk signaling pathways. although further studies of the antiviral effects 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modifications enhance the anti-influenza a virus activities of novel quindoline derivatives antiviral potential of erk/mapk and pi k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade receptor tyrosine kinase inhibitors block multiple steps of influenza a virus replication inhibition of influenza virus-induced nf-kappab and raf/ mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo a new player in a deadly game: influenza viruses and the pi k/akt signalling pathway a central role for pi k-akt signaling pathway in linking samhd -deficiency to the type i interferon signature animal models for the study of influenza pathogenesis and therapy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions ww and ch wrote the manuscript and designed the experiments. yjy, yz, sw and wl performed the experiments. ww, yy and yz analyzed the data. all authors read and approved the final manuscript. this work was supported by national natural science foundation of china ( , , and ), nsfc-shandong joint fund (u , u ), and shandong provincial natural science foundation (zr mh ). the datasets used during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate this work was approved by the ethics committee of ocean university of china. no applicable. the authors declare that they have no competing interests. key: cord- -dwjijmwz authors: volmer, romain; mazel-sanchez, beryl; volmer, christelle; soubies, sébastien m; guérin, jean-luc title: nucleolar localization of influenza a ns : striking differences between mammalian and avian cells date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: dwjijmwz in mammalian cells, nucleolar localization of influenza a ns requires the presence of a c-terminal nucleolar localization signal. this nucleolar localization signal is present only in certain strains of influenza a viruses. therefore, only certain ns accumulate in the nucleolus of mammalian cells. in contrast, we show that all ns tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described c-terminal nucleolar localization signal. thus, nucleolar localization of ns in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains. the nucleolus is a highly dynamic multifunctional subnuclear compartment [ ] . it is the site of ribosomal rna synthesis and ribosomal subunits assembly. in addition, the nucleolus is increasingly recognized as a critical regulator of many other cellular functions, including the regulation of mitosis, cell growth and response to stress [ ] [ ] [ ] . the nucleolus is also emerging as an important target of various viral proteins [ ] . viral proteins targeting the nucleolus are for example implicated in the regulation of apoptosis, as shown with west nile virus capsid protein, and in the regulation of viral mrna export, as shown with human immunodeficiency virus rev protein and with herpesvirus saimiri orf protein [ ] [ ] [ ] . however, for most viruses, consequences of viral protein localization in the nucleolus remain largely unknown [ , ] . the non-structural (ns ) protein of influenza a viruses ns is a multifunctional protein, known to interact with and modify the function of many cellular proteins, thereby creating a cellular environment favouring virus replication [ ] . recently, a nucleolar localization signal (nols) has been identified in ns [ ] . this nols targets ns to the nucleolus of mammalian cells. presently, the role of the nucleolar localization of ns in the viral cycle is unknown. one can speculate that ns proteins targeting the nucleolus of mammalian cells could modify the functions of nucleolar proteins. the mammalian nols of ns consists of a stretch of c-terminal basic amino acids that are present only in certain strains of influenza a viruses [ ] . thus, only certain ns proteins accumulate in the nucleolus of mammalian cells. whether ns proteins accumulate in the nucleolus of avian cells is currently unknown. in this study, we compared the nucleolar localization of ns of different influenza virus strains in mammalian and avian cells using immunocytochemistry and confocal microscopy. experiments were done in human a alveolar epithelial cells and in primary embryonic fibroblasts used between passages and , cultured from days old balb/c mouse (mus musculus) embryos, from days old pekin duck (anas platyrhynchos) embryos or from days old chicken (gallus gallus) embryos. cells were infected at a multiplicity of infection (moi) of plaque forming units (pfu) per cell (moi = ) with the human influenza a/udorn/ (h n ) strain (designated udorn), the human laboratory adapted influenza a/pr/ / (h n ) strain (designated pr ), the avian influenza a/turkey/italy/ /v (h n ) strain (designated ) or the avian influenza a/turkey/italy/ / (h n ) strain (designated ). at , , , and hours post-infection (hpi), cells were fixed with % paraformaldehyde, permeabilized with phosphate buffered saline (pbs) . % triton x- and incubated for one hour in pbs . % triton x- and % bovine serum albumin. antibody incubation was performed overnight at °c. the c-terminal sequence of udorn ns protein contains the basic amino acids identified by melen et al. as * correspondence: rvolmer @gmail.com defining the mammalian nols (underlined in figure ), whereas the other ns proteins lack one or more of these basic amino acids [ ] . consequently, only the ns of udorn accumulated in the nucleolus of primary mouse embryonic fibroblasts (mef) and of a human respiratory cells (figure ). ns proteins of the other viruses tested did not accumulate in the nucleolus of mammalian cells irrespective of the time post-infection ( figure ). by contrast, the ns of all viruses used in this study accumulated in the nucleolus of primary duck embryonic fibroblasts (def) and primary chicken embryonic fibroblasts (cef) at hpi ( figure ). thus, all ns proteins tested have an amino acid sequence forming a functional nols in avian cells. in addition, our results show that the amino acids required to target ns to the nucleolus of avian cells differ from the amino acids required to target ns to the nucleolus of mammalian cells. then, we verified that ns targets the nucleolus in vivo. we infected two-week old pekin ducks orally with (figure ) revealed the presence of viral antigens in enterocytes days post-infection. anti-ns antibodies detected with a peroxidase-coupled secondary antibody revealed with diaminobenzidine stained the cytoplasm and subnuclear structures, corresponding to nucleoli ( figure ) . thus, the subcellular localization of ns in vitro is consistent with its nucleolar localization in duck intestinal epithelial cells. viral infections can lead to changes in the nucleolar morphology, likely caused by virus-induced disruption of nucleolar functions, as shown for the infectious bronchitis coronavirus and for the herpes simplex virus [ , ] . we therefore analyzed whether nucleolar localization of ns modified the expression pattern of nucleophosmin (npm), a nucleolar protein that localizes to the granular centre of the nucleolus [ ] . we performed a time course analysis of the intracellular localization of ns and npm (figure ) in def infected at a moi = with either the or the udorn viruses. in def, ns of both viruses colocalized with npm ( figure ). nucleolar localization of ns was visible hpi and was maximal between and hpi ( figure ). nucleolar accumulation declined starting hpi. the intensity of nucleolar ns staining eventually became indistinguishable from the nucleoplasmic ns staining between and hpi. in addition, we detected bright cytoplasmic foci of ns in def infected with the virus ( figure & ). these foci were reminiscent of previously described virus-induced cytoplasmic inclusions that remain of uncertain identity [ ] . no apparent change in the pattern of npm expression was observed in def infected with the virus. by contrast, starting hpi, faint npm staining could be detected in the nucleoplasm of udorn infected cells, suggesting that a fraction of npm is displaced from the nucleolus to the nucleoplasm following infection. increased levels of npm in the nucleoplasm, as well as ring-like npm staining pattern were detected in about % of udorn infected cells at hpi. interestingly, changes in npm staining pattern has also been observed following infection with the coronavirus infectious bronchitis virus whose nucleocapsid protein targets the nucleolus [ ] . in def infected with udorn, changes in the nucleolar morphology appeared between and hpi, corresponding to a stage in the virus life cycle where cytopathic effects, such as membrane blebbing became visible (date not shown). thus, rather than being due to a direct effect of ns on nucleolar functions, disruption of the nucleolar morphology in influenza virus infected cells could result from virus induced intracellular stress. alternatively, displacement of npm from the nucleolus to the nucleoplasm could be due to an interaction of the viral ribonucleoprotein complex with npm, as shown in mdck cells infected with the influenza a/wsn/ virus [ ] . presently the role of the nucleolar localization of ns in influenza virus cycle is unknown. in mammalian cells, nucleolar accumulation of ns occurs only with certain strains of influenza a viruses. as the ns of all viruses studied here targeted the nucleolus of avian cell, we speculate that the nucleolar localization of ns could be an important step during the viral cycle in avian cells. whether, nucleolar localization of ns contributes to virulence is currently unknown. valuable information would certainly be obtained by studying the phenotype of a reverse genetics engineered virus lacking a functional nols. in order to perform such studies in avian cells, the avian nols needs to be identified. our results show that the avian nols relies on an amino acid sequence that is present in all the influenza virus strains tested in this study, and thus could be conserved among most influenza virus strains. the multifunctional nucleolus cellular stress and nucleolar function nucleolus: the fascinating nuclear body rna viruses: hijacking the dynamic nucleolus nucleolar trafficking is essential for nuclear export of intronless herpesvirus mrna west nile virus capsid protein induces p -mediated apoptosis via the sequestration of hdm to the nucleolus nucleoporins nup and nup participate in nuclear export of human immunodeficiency virus type rev the multifunctional ns protein of influenza a viruses nuclear and nucleolar targeting of influenza a virus ns protein: striking differences between different virus subtypes changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus involvement of ul in herpes-simplexvirus- -induced dispersal of nucleolin phosphorylation of influenza virus nucleoprotein in vivo identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches nucleolar localization of influenza a ns : striking differences between mammalian and avian cells we thank i. capua and w. dundon (istituto zooprofilattico sperimentale delle venezie, legnaro, italy), n. naffakh (institut pasteur, paris) and r. fouchier (erasmus university, rotterdam, netherlands) for the kind gift of viruses, d. marc (inra, tours, france), j. ortin (centro nacional de biotecnología, madrid, spain) for the kind gift of antibodies. the authors declare that they have no competing interests. key: cord- -wy c o authors: kong, ning; meng, qiong; jiao, yajuan; wu, yongguang; zuo, yewen; wang, hua; sun, dage; dong, sujie; zhai, huanjie; tong, wu; zheng, hao; yu, hai; tong, guangzhi; xu, yongjie; shan, tongling title: identification of a novel b-cell epitope in the spike protein of porcine epidemic diarrhea virus date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: wy c o background: porcine epidemic diarrhea virus (pedv) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. during pedv infection, the spike protein (s) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. however, the neutralizing b-cell epitopes within pedv s protein have not been well studied. methods: to accurately identify the important immunodominant region of s , the purified truncated s proteins (sa, sb, sc, sd and se) were used to immunize balb/c mice to prepare polyclonal antibodies. the antisera titers were determined by indirect elisa, western blot and ifa after four immunizations to find the important immunodominant region of s , and then purified the immunodominant region of s protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the b-cell epitopes of monoclonal antibodies. results: five antisera of recombinant proteins of the spike protein region of pedv were generated and we found that only the polyclonal antibody against part of the s region (signed as se protein, residues – ) could recognize the native pedv. purified se protein was used to immunize balb/c mice and generate mab e . pepscan of the se protein demonstrated that se (( )sstfnstrel( )) is the minimal linear epitope required for reactivity with the mab e . further investigation indicated that the epitope se was localized on the surface of pedv s protein in the d structure. conclusions: a mab e that is specifically bound to pedv was generated and identified a specific linear b-cell epitope (se , ( )sstfnstrel( )) of the mab. the epitope region of pedv s localized in the different regions in comparison with the earlier identified epitopes. these findings enhance the understanding of the pedv spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect pedv. porcine epidemic diarrhea (ped) is an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets [ , ] . the disease was reported in european and asian pig industries over the last years, with the virus firstly appearing in england and belgium in the early s [ , ] . porcine epidemic diarrhea virus (pedv), although the etiologic agent of ped has become a severe problem in many asian countries, including china, korea, japan and thailand [ ] [ ] [ ] [ ] . since , the virulent pedv has become prevalent in swine herds and incurred huge economic losses to the swine industry [ ] [ ] [ ] . due to the lack of effective vaccines, ped is still circulating in the worldwide. pedv belonging to the genus alphacoronavirus, family coronaviridae, has an approximately kb genome of single-stranded, positive-sense rna [ ] . the pedv genome encodes two large polyproteins, an accessory protein and four structural proteins. the structural proteins contain glycosylated spike (s), envelope (e), glycosylated membrane (m) and rna-binding nucleocapsid (n) proteins [ ] . the spike gene can be divided into s and s domains, as in other coronaviruses and it has multiple functions that can interact with cellular receptors and regulating viral entry and containing neutralizing epitopes to induce neutralizing antibodies [ , ] . in the present study, we expressed and purified the recombinant truncated pedv s constructs (sa-se) to immunize balb/c mice and found that se, one of the s construct (residues - ), was the immunodominant region of s protein. furthermore, we utilized the se protein to immunize balb/c mice and obtained one se specific mab, e . a novel linear b-cell epitope, ( sstfnstrel ), was subsequently identified using the se specific mab e . these results provide valuable information for virus diagnosis and vaccine design. african green monkey kidney cells (vero e ) and sp / myeloma cells were cultured in a humidified % co atmosphere at °c. all the culture media were dulbecco's modified eagle's medium (dmem, hyclone) supplemented with % fetal bovine serum (fbs) and antibiotics ( . mg/ ml of streptomycin and iu/ml of penicillin). pedv strain js- was obtained from the shanghai veterinary research institute (caas, china). plasmid dna (pcold-tf) containing the s gene ( - bp) of pedv strain js- was constructed by our own laboratory. the five overlapping fragments, comprising partial length of pedv s gene, were constructed and designated as sa, sb, sc, sd and se. a bamh i site and sixteen extra bases that were homologous to the terminal sequence of the vector were added to the ′ end. the sequences of the primers used for amplification of the gene in this study are shown in table . all the recombinant plasmids were constructed by clonexpress ii one step cloning kit (vazyme biotech, c - ), according to the manufacturer's instructions. the truncated segments sa-se were cloned into pcold-tf vector and the confirmed recombinant plasmids were transformed into e. coli bl and induced by isopropyl-β-d-thiogalactoside (iptg) at °c for h. the truncated pedv s proteins were analyzed by sodium dodecylsulfatepolyacrylamide gel electrophoresis (sds-page) and western blot. all the recombinant proteins were purified by using nickel magnetic beads (biotool, shanghai, china) to prepare polyclonal antibodies. groups of five -week-old female balb/c mice were intraperitoneally immunized with μg purified truncated pedv s proteins. antigens were emulsified in the same volume of complete freund's adjuvant (sigma, usa) for the initial immunization, then emulsified in incomplete freund's adjuvant on subsequent immunizations at week intervals for weeks. phosphate-buffered saline (pbs) was used for the controlled trial with the same procedures. three days after the final boosting, the mice were narcotized and their blood samples were collected from the caudal vein. the collected antisera were diluted -fold and used for indirect elisa, western blot and ifa to detect the immunoactivity of truncated proteins. female -week-old balb/c mice were immunized with μg purified protein emulsified in the same volume of complete freund's adjuvant via intraperitoneal injection. this procedure was followed by three additional injections at -week intervals with the same dose of antigen emulsified in incomplete freund's adjuvant. three days after the final injection, spleen cells from immunized mice were fused with sp / myeloma cells using polyethylene glycol (peg , sigma, usa), as previously described [ ] . then the hybridoma cells were seeded into -well plates and selected in hypoxanthine-aminopterin-thymidine (hat) medium and hypoxanthine-thymidine (ht) medium. the cell culture supernatants of surviving clones were determined by indirect elisa for antibody reactivity and specificity. positive hybridomas were cloned four times by limiting dilution. ascites fluids were produced in pristane induced balb/c mice. indirect elisa was used to identify the immune reactivity of the truncated proteins and the screen of positive hybridoma cells. the elisa plates were plated with purified pedv s protein or synthesized peptides ( ng/well) in carbonate bicarbonate buffer ( mm na co , mm nahco [ph . ]) and coated at °c overnight. the plates were blocked for h at °c using % non-fat dry milk in phosphate buffer with . % tween- (pbst). after being washed thrice, the plates were incubated with μl diluted anti-sera or antibodies at °c for h. the plates were incubated with horseradish peroxidase (hrp) -conjugated goat anti-mouse igg (proteintech group, china) with : , dilution in pbst at °c for h after being washed thrice in pbst. then, plates were washed with pbst and incubated with μl/well of tmb liquid (amresco, solon, ohio, usa) for min at room temperature with protection from light. the results were read with od values after being stopped by m h so ( μl/well). to analyze the anti-sera or antibodies specificity interacted with pedv, vero cells were infected with pedv (multiplicity of infection, moi = ), or mock infected with the medium, and then incubated for indicated times as previously described [ ] . the cells were harvested and lysed using ripa lysis buffer (thermo, usa) containing protease inhibitor cocktail (bimake, usa) and phosphatase inhibitor cocktail (bimake, usa) on ice for min. the cell lysates were then separated by % sds-page and transferred to the nitrocellulose (nc) membrane (ge healthcare, usa). the membranes were blocked with % non-fat dry milk in tbst (tbs with . % polysorbate- ) for h at room temperature (rt). the membranes were subsequently incubated with hrp-conjugated goat anti-mouse igg ( : dilution in tbst) for h at rt. proteins were visualized by using supersignal west pico chemiluminescent substrate (thermo fisher scientific, usa) according to the manufacturer's instructions. vero cells were plated in a six-well plate and infected with pedv when the cells reached approximately % confluence. at h postinfection, the cells were fixed with % paraformaldehyde (sigma-aldrich) for min and permeabilized with . % triton x- (sigma-aldrich) for min at room temperature. after being washed three times in phosphate-buffered saline (pbs), the cells were blocked with % bovine serum albumin (bsa) in pbs for h at °c and then incubated with the primary antibody for h. after three washes with pbs, cells were incubated with alexa fluor donkey anti-mouse igg (h + l) antibody in the dark for h at °c. following several washes, the fluorescence was visualized by using an olympus® ix inverted microscope. the spatial position of the identified epitope was analyzed by mapping the location on the d structure model of pedv s [ ] by using pymol software [ , ] , and the secondary structure of amino acid sequences of the identified epitope was also analyzed by protean software (dnastar's lasergene, inc., madison, wi, usa) [ ] . all results are representative of three independent experiments. statistical analysis was performed using prism . software (graphpad). significance was determined by two-tailed student's t test. statistical significance: *p < . , **p < . . immunodominant region of s protein to accurately identify the immunodominant region of s , the purified truncated s proteins (sa, sb, sc, sd and se) were used to immunize balb/c mice to prepare fig. detection of recombinant proteins' antigenicity for polyclonal antisera (pcabs) by indirect elisa, western blot and ifa. a reactivity of antisera against the recombinant s protein by indirect elisa. b western blot analysis of the recombinant s protein with pcabs. the recombinant s protein (approximately kda) was transferred to the nitrocellulose (nc) membrane, followed by the different pcabs (negative control, sa polyclonal antisera, sb polyclonal antisera, sc polyclonal antisera, sd polyclonal antisera and se polyclonal antisera) as primary antibody. c immunofluorescence analysis of se polyclonal antisera against pedv. vero cells were plated in six-well plates and inoculated with pedv ( . moi). twenty-four hours later, cells were fixed and incubated with se polyclonal antisera or normal mouse serum (negative control), and then incubated with alexa fluor donkey anti-mouse igg (h + l) antibody. scale bars: μm polyclonal antibodies. the antisera titers were determined by indirect elisa, western blot and ifa after four immunizations. the indirect elisa and western blot analysis showed that the se polyclonal antisera had the highest antibody titers against pedv s protein ( fig. a and b) , suggesting that se protein was the important immunodominant region of s . the immunofluorescence signal for the pedv s protein was also detected by the se polyclonal antisera (fig. c) , further confirming that se polyclonal antisera could recognize the native pedv. collectively, these results indicated that the region of se ( - aa) was the immunodominant region of s protein. purified se protein was used to immunize balb/c mice to prepare mabs, and then determined the antisera titers using indirect elisa after four immunizations. the mouse with the highest antibody titers against se protein was used for cell fusion. after being subcloned by limiting dilution and screening for four times, one positive mab against se protein was identified and named e . the mab e cell clone was used to prepare ascites containing mabs. the ascites was collected and purified using nab protein g spin columns (thermo fisher scientific, rockford, il). western blot analysis (fig. a) and immunofluorescence assay (ifa) (fig. b) were used to identify the specificity of the mabs against pedv. the results suggested that the mab e could be specifically reacted with native pedv protein. to identify the antigenic epitope recognized by mab e , four truncated and overlapping × his-tagged peptides (se -se ) spanning the se fragments were designed (fig. a) and expressed using the bacterial system. the sequences of the primers were also shown in table . all fusion proteins were predominantly expressed in soluble form in bacterial cells. subsequently, western blot was used to determine immune reactivity between the mab e and these se fragments. the results showed that mab e reacted with fragments spanning aa to (se ) and to (se ) but not aa to (se ) or to (se ), suggesting that the epitope recognized by e was located in aa to of pedv s protein (fig. b) . furthermore, we generated two deletion constructs of se (se , se ) that span the overlaps of se and se (fig. a) to identify the antigenic epitope. as demonstrated by western blot, se was recognized by mab e (fig. c) , which suggested that se ( isslssstfnstrelpgffy ) epitope may be a harbored antigenic epitope. in order to further minimize the epitope of se , two shortened peptides (se -se ) were synthesized via solid-phase peptide synthesis (table ) . using indirect elisa, we found that se showed a strong reaction with mab e as se did (fig. a) . the result suggested that se was the essential region for recognition by mab e . according to the results, four shortened peptides (se -se ) by deleting three amino acids at either the amino or the carboxy terminus in sequence from the se were synthesized ( table ). in elisa, only se could be recognized by mab e , which indicates that the e -specific epitope was sstfnstrelpg (fig. a) . with the same method, the other four shortened peptides (se -se ) were synthesized according to the peptide sequence of se . the results showed that the peptide se ( sstfnstrel ) was strongly recognized by the mab e (fig. a) . taken together, these results demonstrate that se ( sstfnstrel ) is the minimal linear epitope required for reaction with the mab e . to localize the identified epitope se , a d structural model of pedv s was obtained from the protein data bank (pdb, id: u k), and the spatial distribution was analyzed by pymol software. the structural visualization revealed that the identified epitope recognized by e was exposed on the surface of pedv s structure (fig. b) and partial peptide formed a beta-sheet structure (fig. c) . moreover, the identified epitope se had high antigenic index and hydrophilicity (fig. d) . it was suggested that the epitope can easily explore and induce host immune response in those infected animals. pedv s protein, one of the most important glycoproteins, contains multiple neutralizing epitopes to induce neutralizing antibodies. the s protein is divided into s (residues - ) and s (residues - ) domains which are defined by the conserved nonamer and the gxcx motifs in coronavirus group ii members [ ] . based on the information of other coronaviruses, we found that the s domain is thought to contain multiple virus neutralization epitopes and receptor bindingdomains [ ] . the s domain forms the trans-membrane structure of the s protein, but can not induce neutralizing antibodies [ ] . these properties make it possible that the s could be a suitable candidate for screening and identifying antigenic epitopes. epitopes are important antigenic elements of virus structural proteins, which could induce antibody production and cell-mediated immunity against viruses. therefore, epitopes are essential to develop epitope-based vaccines and diagnostics. preparation of mab is required to identify the epitopes of the s protein. we had tried to use intact s protein to immunize mice, but it was unsuccessful to get the mice serum with immune activity. because of unidentified but complex factors, we chose to divide the s protein into five fragments (sa, sb, sc, sd, se). the five truncated proteins were expressed and the immunogenicity of the proteins was identified. these results indicated that the se protein ( - aa) had good reactivity as the immunodominant region of s protein. then the se protein was selected as an immunogen to elicit the formation of monoclonal antibody. after cell fusion and four times of selection, mab e was chosen because of its specific reactivity with the se protein as well as the native s protein pedv. several domains containing neutralizing epitopes within the s protein were identified, such as residues - [ ] , residues - [ ] , residues - [ ] and residues - [ ] . these mocking epitopes had antigenic similarities with the pedv neutralizing epitopes. in this study, we expressed a series of truncated proteins (se -se ) to map the epitopes of se protein. the se protein could be recognized by mab e . generally, linear epitopes consist of six to nine or more continuous amino acid residues. so, the pepscan method was used to fig. pepscan of the epitopes se and localization of the epitopes recognized by the mab e . a elisa analysis of the truncated peptides se -se with mab e and pbs. the surface b and cartoon c from se was labeled in the sequence chain view picture of pedv s, obtained from the protein data bank (pdb, id: u k). d the structural features of se were predicted by protean software. the epitope se was shown in the boxes truncate the epitope se by deleting three amino acids at either the amino or the carboxy terminus in sequence respectively. a total of ten sub-segments (se -se ) was synthesized and the results of elisa suggested that se could react with mab e . above all these results, the epitope se is the immunodominant region of pedv s protein. the epitope se ( sstfnstrel ) was highly conserved among different strains of virulent pedv. further, the d structural visualization showed that the epitope se presented on the surface of pedv s and had a feature of high antigenic index and hydrophilicity. the character of this location makes this epitope easily explore and induce host immune response in the infected animals. we prepared a mab e that is specifically bound to pedv and identified a specific linear b-cell epitope (se , sstfnstrel ) of the mab. the epitope region of pedv s localized in the different regions in comparison with the earlier identified epitopes. therefore, the identified region is a novel b-cell antigenic epitope region of pedv s protein and the identified epitope has potential use for developing diagnostic reagent and effective vaccines for pedv. a new coronavirus-like particle associated with diarrhea in swine threedimensional sequential study of the intestinal surface in experimental porcine cv coronavirus enteritis virus-like particles associated with porcine epidemic diarrhoea an apparently new syndrome of porcine epidemic diarrhoea chinese-like strain of 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range identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification of a novel linear b-cell epitope within the collagenase equivalent domain of porcine epidemic diarrhea virus spike glycoprotein identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank mr. yongjun chen for his assistance in animal feeding. availability of data and materials data generated and analyzed in this study are presented in this manuscript. data can be obtained by contacting the corresponding author. the authors declare that they have no competing interests.author details key: cord- -xcwppaux authors: esposito, susanna; molteni, claudio g; daleno, cristina; valzano, antonia; tagliabue, claudia; galeone, carlotta; milani, gregorio; fossali, emilio; marchisio, paola; principi, nicola title: collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: xcwppaux this study evaluated the efficiency of pediatric mid-turbinate nasal flocked swabs used by parents in children aged months to years with signs and symptoms of respiratory disease. two nasal samples were collected from each child in a randomised sequence: one by a trained pediatrician and one by a parent. the real-time polymerase chain reaction influenza virus detection rates were similar in the samples collected using the two methods (cohen's kappa = . ), as were the cycle threshold values. in comparison with the pediatrician-collected samples, the sensitivity and specificity of the parental collections were respectively . % ( % confidence interval [ci]: . - %) and . % ( % ci: . - %), and the positive and negative predictive values were respectively . % ( % ci: . - . %) and . % ( % ci: . - %). the children were significantly more satisfied with the parental collections (median values ± standard deviation, . ± . vs . ± . ; p < . ). these findings show that mid-turbinate nasal flocked swabs specifically designed for infants and children can be used by parents without reducing the influenza virus detection rate. moreover, the direct involvement of parents significantly increases patient acceptance, thus simplifying collection and suggesting that this novel swab design should be considered for epidemiological surveys and vaccine efficacy studies. in order to monitor the circulation of infectious agents and evaluate the efficacy of specific vaccines, it is essential to be able to identify the viruses that cause respiratory diseases in infants and children [ ] [ ] [ ] [ ] [ ] [ ] , and the adequate collection of respiratory specimens is the first crucial step in obtaining reliable information [ ] [ ] [ ] . such specimens are usually collected in hospital by certified nurses, pediatricians or other medical doctors, but parents may find it troublesome having to go to a hospital every time a specimen needs to be taken from a child with respiratory infection as such diseases occur several times a year. collecting respiratory secretions at home could overcome this, but traditional collection techniques (mainly nasopharyngeal aspiration and nasopharyngeal washing) are too complex, invasive and time-consuming to be used by untrained people [ ] [ ] [ ] . it has been found that recently developed mid-turbinate nasal flocked swabs are as effective as these traditional methods [ ] [ ] [ ] , and simple enough to be used by adult patients themselves and the parents of children [ , ] . however, as experience with the parental collection of samples is very limited, we evaluated the efficiency of pediatric mid-turbinate nasal flocked swabs when used by parents. the study involved all of the children aged between six months and five years who attended the emergency department of the university of milan's department of maternal and pediatric sciences because of signs and symptoms of respiratory disease between january and february . only the children with known craniofacial abnormalities were excluded. the protocol was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, and two nasal samples were collected in a randomised sequence from each child: one by a trained pediatrician (ct) and one by a parent. the pediatric mid-turbinated nasal flocked swabs (copan, brescia, italy, code cs , suitable for children aged up to two years) and those for older children (code cs ) have a collar respectively . and . centimeters along the swab shaft ( figure ) that is large enough to prevent further insertion when it reaches the nostril. the pediatrician and a parent (who was first asked to read a very simply written and illustrated description of the procedure) each inserted a swab gently up to its collar and rotated it three times before placing it in viral transport medium to be delivered to the laboratory within three hours. the parents were then asked to describe their child's satisfaction with the two procedures using a a five-point scale (from for "very satisfied" to for "very unsatisfied"); an independent observer (pm) confirmed that the child's satisfaction was as reported by the parents. there was no refusal to participate, all of the children had two swabs taken (one by the pediatrician and one by a parent), and a satisfaction scale was completed for each. as soon as they were delivered to the laboratory, each patient's paired samples were processed in parallel. viral rna was extracted from both swabs by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), using phocine distemper virus (pdv) as an extraction control as previously described [ , ] . all of the real-time polymerase chain reactions (pcrs) were set up as singleplex pcrs in a total volume of μl, using the taqman universal master mix (applied biosystems, foster city, ca, usa), - nm of primers, nm of taqman probe and μl of cdna template, and the products were amplified using the abi ht fast real-time pcr system (applied biosystems) and standard cycling parameters. the primerprobe sets were: influenza a, sense aagaccaatcct-gtcacctctga, antisense caaagcgtctacgctg-cagtcc, probe fam-tttgtgttcacgctc acc gtgcc-bhq ; influenza b, sense gagacacaattgc-ctacctgctt, antisense ttctttcccaccga accaac, probe tet-agaagatggagaagg caaag cagaactagc-eclipse; pdv, sense cgggtgccttt-tacaagaac, antisense ttctttcctca acctcg tcc, probe vic-atgcaagggccaattcttccaag tt-bhq . influenza a and b rna were quantified relatively; the criterion for a positive reaction was a cycle threshold (ct) of < cycles. the findings relating to the specimens collected by the parents and pediatricians were compared using sas ver- the categorical data were compared between groups using the χ test or fisher's test; the other between-group comparisons were made using wilcoxon's signed-rank test, a non-parametric test for paired samples. p values of . or less were considered statistically significant. the mean age ± standard deviation (sd) of the recruited children was . ± . years: ( . %) were younger than two years, and the specimens were taken using the smaller swabs. table shows the detected influenza viruses. thirty-two children ( . %) were positive for influenza: the paired samples were both positive in cases ( . %), only the samples collected by the pediatrician were positive in three cases ( . %), and only the samples collected by a parent were positive in four cases ( . %). the influenza virus detection rates were similar in the samples collected using the two methods (cohen's kappa = . ): in comparison with the pediatricians, the sensitivity and specificity of the parental collections were respectively . % ( % confidence interval [ci]: . - %) and . % ( % ci: . - %), and the positive and negative predictive values were respectively . % ( % ci: . - . %) and . % ( % ci: . - %). table summarises the ct values in the paired positive samples, which show that similar amounts of viruses were detected in the samples collected using the two methods. however, the children were significantly more satisfied with the parental collections (mean values ± sd, . ± . vs . ± . ; p < . ). the detection and satisfaction rates were similar regardless of the patients' age. our findings demonstrate that mid-turbinate nasal flocked swabs specifically designed for infants and children can be used by parents without reducing influenza virus detection rates. the number of influenza-positive nasal swabs and the ct values were similar in the samples collected by the pediatrician and parents. furthermore, the direct involvement of parents significantly increased the patient's acceptance of the procedure and thus simplified collection. these results suggest that, when an early evaluation of the viral etiology of a respiratory tract infection is needed, parents can collect respiratory secretions at home using pediatric mid-turbinate nasal flocked swabs. this has a number of advantages. first of all, if the child is included in an epidemiological survey or vaccine efficacy study, parental collection reduces the risk of losing the sample when respiratory episodes occur. secondly, the samples can be obtained immediately after the onset of the first signs and symptoms, thus favouring the identification of the infectious agent and aiding treatment decision making after a pediatrician's visit. thirdly, it reduces family organisational problems and the children's emotional involvement. however, in order to make the most of such advantages, appropriate swabs specifically designed for infants and young children need to be used because the shafts of adult swabs are too long, and their tips are too big. specifically prepared mid-turbinate nasal flocked swabs with a collar that prevents them from being inserted so deeply that they come into possibly painful contact with inflamed structures are safe and well tolerated, and can therefore be recommended in routine practice. we do not know why the paired samples of seven children were not both positive, but only those taken by the pediatrician in three cases, and only those taken by a parent in four. there were no differences in ct values suggesting less virus and lower sensitivity, and no differences in the timing of the collections or in the age or weight of the children. in any case, the detection rates of the two collection methods were similar, and the sensitivity, specificity and positive and negative predictive values were high. one limitation of this study is that, although the parents collected the respiratory secretions without any particular assistance, they were in our hospital and probably felt more confident knowing that professional help was on hand if needed; it is possible that they may have found it more difficult at home or that the sampling would have been less precise. to reduce these risks, it seems reasonable to suggest that they should be instructed by their child's pediatrician and that an illustrated explanation with details concerning specimen storage and transportation should be included in the package insert. moreover, the study population was small and only influenza viruses were evaluated. however, although further studies of larger populations designed to detect other respiratory viruses would strengthen our conclusions, we suggest that this novel swab design would be useful for epidemiological surveys or vaccine efficacy studies. list of abbreviations ct: cycle threshold; ci: confidence interval; pdv: phocine distemper virus; pcr: polymerase chain reaction; sd: standard deviation. the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital rapid assays for the diagnosis of influenza a and b viruses in patients evaluated at a large tertiary care children's hospital during two consecutive winter seasons effect of a rapid influenza diagnosis impact of human coronavirus infections in otherwise healthy children who attended an emergency department the global state of influenza in children impact of human bocavirus on children and their families influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza antigen-based assays for the identification of influenza virus and respiratory syncytial virus: why and how to use them in pediatrics comparison of nasopharyngeal nylon flocked swabs with universal transport medium and rayon bud swabs with a sponge reservoir of viral transport medium in the diagnosis of paediatric influenza nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children comparison of four nasal sampling methods for detection of viral pathogens by rt-pcr-a ga( ) len project comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients comparison between pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time nasba comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood this study was supported in part by a grant from the italian ministry of health (bando giovani ricercatori ). the authors declare that they have no competing interests. se and np designed the study and co-wrote the manuscript. cgm, cd and av carried out the real-time pcr. ct collected the swabs. cg performed the statistical analysis. gm, ef and pm examined the patients. all authors read and approved the final manuscript. key: cord- -roftm authors: holtz, lori r; finkbeiner, stacy r; zhao, guoyan; kirkwood, carl d; girones, rosina; pipas, james m; wang, david title: klassevirus , a previously undescribed member of the family picornaviridae, is globally widespread date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: roftm background: diarrhea is the third leading infectious cause of death worldwide and is estimated to be responsible for approximately million deaths a year. while many infectious causes of diarrhea have been established, approximately % of all diarrhea cases are of unknown etiology. in an effort to identify novel viruses that may be causal agents of diarrhea, we used high throughput mass sequencing to analyze stool samples collected from patients with acute diarrhea. results: sequences with limited similarity to known picornaviruses were detected in a stool sample collected in australia from a child with acute diarrhea. using a combination of mass sequencing, rt-pcr, ' race and ' race, a bp fragment of the viral genome was sequenced. phylogenetic analysis demonstrated that this virus was highly divergent from, but most closely related to, members of the genus kobuvirus. we have tentatively named this novel virus klassevirus . we also detected klassevirus by rt-pcr in a diarrhea specimen collected from a patient in st. louis, united states as well as in untreated sewage collected in barcelona, spain. conclusion: klassevirus is a previously undescribed picornavirus that is globally widespread and present on at least three continents. further investigations to determine whether klassevirus is a human pathogen are needed. the impact of diarrhea is primarily felt in the developing world, where approximately million deaths result from diarrhea annually [ ] [ ] [ ] . in developed countries, where diarrhea related mortality is relatively rare, there is still nonetheless a tremendous disease burden. for example, in the united states, approximately % of all hospitalizations for children under age years are due to diarrhea episodes [ ] . while rotaviruses, caliciviruses, adenoviruses, and astroviruses are responsible for the greatest proportion of cases [ ] [ ] [ ] [ ] , approximately % of diarrhea cases are of unknown etiology [ ] [ ] [ ] . many picornaviruses can be detected in human stool such as enteroviruses, polio, aichi virus, and cardioviruses [ ] [ ] [ ] [ ] . some of these viruses, such as aichi virus, are associated with diarrheal disease [ ] while others such as polio are shed fecally, but manifest pathogenicity in other organ systems. picornaviruses are non-enveloped viruses with a single stranded positive-sense rna genome that encodes a single polyprotein [ ] . the picornavirus family currently consists of proposed genera http://www.picor naviridae.com associated with a diverse range of diseases. viruses in six of these genera potentially infect humans (enterovirus, hepatovirus, parechovirus, kobuvirus, cosavirus, and cardiovirus). with the advent of culture independent molecular methods, many diverse new members of the picornavirus family have been identified in recent years. these include novel cardioviruses [ ] [ ] [ ] [ ] , rhinoviruses [ ] [ ] [ ] , parechoviruses [ , ] and the novel genus of cosaviruses [ , ] . these studies have demonstrated that significant viral diversity exists in the human gut that remains unexplored. we have previously described a mass sequencing strategy based on high throughput sanger sequencing to analyze human stool for previously undescribed viruses [ ] . in this study, we used a similar strategy but incorporated a next-generation pyrosequencing platform (roche genome sequencer) in place of traditional sanger sequencing. this resulted in the identification of a highly divergent picornavirus in a stool sample collected in from a child in australia with acute diarrhea. sequencing and phylogenetic analysis demonstrated that this virus is a novel member of the family picornaviridae. we propose that this virus be named klassevirus (kobu-like virus associated with stool and sewage). extracted nucleic acid from a stool specimen collected in from a child with acute diarrhea was subjected to high throughput mass sequencing using pyroseqencing technology. from the resulting reads, two sequences were identified by blast that had only limited sequence identity to known viruses. one was a bp fragment that upon translation to amino acid sequence had % identity to its closest relative, aichi virus ( b/ c region). the second sequence read of bp had % amino acid identity to the vp region of aichi virus. from these two initial sequences, a bp contig (klasse-mel ) was generated by rt-pcr and multiple ' and ' random amplification of cdna ends (race) reactions. the ' end of this contig aligned to the predicted vp protein at the n-terminus of the polyprotein and extended past the predicted d protein at the c-terminus of the polyprotein to the poly-a tail. the initial assembly was confirmed by sequencing multiple overlapping rt-products spanning the length of the contig to give . x coverage. we were not able to extend the contig further in the ' direction despite performing multiple ' race reactions using different primers with multiple high temperature ( °c) reverse transcriptases (rtth [applied biosystems] and thermoscript [invitrogen]). the klassevirus contig had a genomic organization similar to other picornaviruses ( figure a -c). conserved pfam [ ] motifs characteristic of picornaviruses were present including two picornavirus capsid proteins, rna helicase, c cysteine protease, and rna dependent rna polymerase. phylogenetic analysis of the vp /vp , p , and p regions of the genome demonstrated that this virus sequence is highly divergent from all previously described picornaviruses (figure a -c). the closest relatives of klassevirus appeared to be members of the genus kobuvirus, which includes aichi virus, bovine kobuvirus and porcine kobuvirus. in order to determine the prevalence of klassevirus , two patient cohorts were examined by rt-pcr. in the first cohort, pediatric stool specimens sent to the clinical microbiology lab for bacterial culture at the st. louis children's hospital in st. louis mo, usa were tested. one sample from this cohort was positive by rt-pcr. the amplicon from this patient's virus (designated klasse-stl ) was found to have % identity at the amino acid level and % nucleotide identity to the original klasse-mel contig. screening of stool samples from children with acute diarrhea collected from the royal children's hospital (melbourne, australia) did not yield any additional positive samples. in addition, rt-pcr was performed on concentrated raw sewage collected in barcelona, spain. a pcr product of the expected size was obtained and genomic organization of kobuviruses directly sequenced. this fragment (klasse-bar ) shared % amino acid identity and % nucleotide identity to the original klasse-mel sequence (table ) . to further compare the divergence between the three positive samples of klassevirus , rt-pcr was performed using primers that target the polymerase region ( d) of the genome and the vp /vp region. approximately one kb of additional sequence was generated from klasse-bar and klasse-stl in both of these regions (see methods). pair-wise amino acid identities ranged from %- %, with the greatest degree of sequence conservation in the d region (table ). in this study, we identified a previously undescribed picornavirus present in stool and sewage. phylogenetic analysis demonstrated that this virus is most closely related to other picornaviruses in the genus kobuvirus. based on the criteria established by the picornavirus study group, members of a genus should share > %, > % and > % amino acid identity in p , p and p genome regions respectively [ ] . klassevirus shared only % amino acid identity in the p region and % amino acid identity in the p region to its closest relative, aichi virus. given these observations, and using strictly the percent identity definitions, klassevirus may represent the first member of new picornavirus genus. however, we note that at all loci, bootstrap analysis suggests that klassevirus diverged from an ancestor common to all of the known kobuviruses. thus the formal classification of klassevirus at the genus level is currently uncertain and subject to further discussion per the ictv. subsequent screening by rt-pcr using primers targeting the c region of the genome established that klassevirus -like sequences were present not only in australia, but also in north america and europe. the presence of klassevirus in the united states was determined by the traditional strategy of screening of individual stool samples. in addition, we also examined raw sewage collected in barcelona to see if we could detect klassevirus . sewage represents a pooled meta-sample of literally thousands of individual specimens. known enteric viruses such as adenoviruses [ , ] , noroviruses [ ] astroviruses [ ] , and hepatitis a [ ] have frequently been tested for and detected in sewage by pcr and rt-pcr. we reasoned that detection of klassevirus in raw sewage would serve as a proxy for its presence in human stool in the population that generated the sewage. since the exact history of the sewage is poorly defined, it is possible that other waste products, such as animal feces could contribute to the raw sewage meta-genome. nonetheless, we propose that raw sewage screening from a diversity of sites can serve to rapidly define the geographic distribution of a given virus. the detection of klassevirus in stool and sewage from melbourne, barcelona and st. louis, demonstrates that klassevirus is globally distributed. moreover, since both the barcelona sewage and st. louis stool specimens were collected in , we conclude that klassevirus is currently circulating in the human population. whether klassevirus represents a true human pathogen remains to be determined. it is possible that klassevirus is a human pathogen that causes gastroenteritis. it is also possible that klassevirus injures other organs but is excreted through the intestinal tract like poliovirus. another possibility is that klassevirus is a human commensal virus. alternatively, klassevirus could represent a non-human virus acquired from dietary exposure. further investigations are needed to determine if klassevirus is a causal agent of human disease(s). to begin addressing this question, epidemiologic studies including case-control and seroprevalence analyses are needed. this stool was collected in from a month old child presenting to the emergency department of the royal children's hospital, melbourne, australia with acute diarrhea and stored at - °c. previous testing of this diarrhea specimen for known enteric pathogens using routine enzyme immunoassays (eia) and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens was negative [ ] . additionally, rt-pcr assays for caliciviruses and astroviruses were also negative [ , ] . sample preparation for sequencing mg of frozen stool was chipped and then resuspended in volumes of pbs [ ] . the sample was centrifuged to pellet particulate matter and the supernatant was then passed through a . μm filter. total nucleic acid was isolated from μl primary stool filtrate using qiamp dna extraction kit (qiagen) according to manufacturer's instructions. total nucleic acid was randomly amplified using the round ab protocol as previously described [ ] . this was then pyrosequenced on a roche flx genome sequencer (roche) according to manufacturer's protocol. to eliminate sequence redundancy in each library sequences were clustered using blastclust from the . . version of ncbi blast. sequences were clustered based on % identity over % sequence length and the longest sequence from each cluster was chosen as the representative sequence of the cluster. unique sequences were filtered for repetitive sequences and then compared with the genbank nr database by blastn and tblastx. for sequencing experiments, the stool filtrate was proteinase k treated prior to rna extraction. rna was isolated from primary stool filtrate using rna-bee (tel-test, inc.) according to manufacturer's instructions. rt-pcr and 'race reactions were performed using superscript iii and platinum taq (invitrogen one-step rt-pcr). for 'race reactions cdna was generated with themoscript (invitrogen) and amplified with accuprime taq (invitrogen). amplicons were either cloned into pcr (invitrogen) or sequenced directly. protein sequences associated with the following reference virus genomes were obtained from genbank: equine ) . the amino acid alignments generated by clustalx were input into paup [ ] , and maximum parsimony analysis was performed using the default settings with , replicates. stool samples were collected from children under the age of who were admitted to the royal children's hospital, melbourne, victoria, australia with acute diarrhea between and . for a portion of these samples ( ), rna was extracted in the same manner as the primary sample. for the remaining specimens ( ), chips of frozen fecal specimens (~ - mg) were resuspended in volumes of pbs. total nucleic acid was extracted from μl of each stool suspension using a magnapure lc instrument (roche). μl of water was used to elute the total nucleic acid from each sample. leftover material from stool specimens that were routinely submitted to the st. louis children's hospital lab for bacterial culture were collected from january -july . for these specimens total nucleic acid was extracted as described above. this study was approved by the human research protection office of washington university. one l-sample of raw sewage was collected in an urban wastewater treatment plant in the area of barcelona, spain. the sample was collected in a sterile container and stored for up to hours at °c before being processed. the viruses present in the sample were concentrated in ml of phosphate buffer by organic flocculation based on the procedure previously described by calgua et al., [ ] . a second concentration step with elution of the viral particles was performed. briefly, ml of the viral concentrate were eluted with ml of . m glycine buffer (ph , ) at °c, suspended solids were separated by low speed centrifugation at × g for min at °c and the viruses present in the supernatant were finally concentrated in ml of pbs by ultracentrifugation at × g for h at °c. this was then dnase treated, and then total nucleic acid was extracted. primers expected to generate a bp product were designed to the c region of klassevirus (lg : '-cgtcagggtgttcgtgatta- ' and lg : '-agaga-gagctgtggagtaattagta- '). rt-pcr reactions were performed using qiagen one-step kit under the following conditions: min rt step, °c hold for min, followed by cycles of °c for s, °c for s, and °c for s. in order to further compare strain divergence, primers expected to produce amplicons of bp and bp based on the klasse-mel sequence were designed targeting the d and vp /vp regions, respectively: (lg : '-atggcaaccctgtccctgag- ' and lg '-ggaaacccaaccacgctgta- ') and (lg : '-gctaactctaatgctgccacc- ' and lg : '-gctaggtcagtggaaggatca- '). these rt-pcr reactions were performed using the invitrogen one-step rt-pcr kit with the following conditions: min rt step at °c, °c hold for min, followed by cycles of °c for s, °c for s, °c for s. whenever possible, amplicons were cloned into pcr (invitrogen) and sequenced using standard sanger sequencing technology. in some instances, pcr products were directly sequenced and only high quality sequence from those samples were included in analysis. all klassevirus sequences have been deposited in genbank (gq -gq ). publish with bio med central and every scientist can read your work free of charge the global burden of diarrhoeal disease a millennium update on pediatric diarrheal illness in the developing world hospital use by pediatric patients: implications for change a review of viral gastroenteritis a -year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in diarrhea etiology in a children's hospital emergency department: a prospective cohort study viruses causing gastroenteritis explaining unexplained diarrhea and associating risks and infections viral gastroenteritis 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genomic characterization of novel human parechovirus type a highly prevalent and genetically diversified picornaviridae genus in south asian children identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea metagenomic analysis of human diarrhea: viral detection and discovery pfam: clans, web tools and services virus taxonomy eighth report of the international committee on taxonomy of viruses edited by: fauquet cm detection of adenoviruses and enteroviruses in polluted waters by nested pcr amplification viral pollution in the environment and in shellfish: human adenovirus detection by pcr as an index of human viruses molecular detection of norwalk-like caliciviruses in sewage astrovirus detection in wastewater samples nested multiplex pcr assay for detection of human enteric viruses in shellfish and sewage improved sensitivity of astrovirus-specific rt-pcr following culture of stool samples in caco- cells viral discovery and sequence recovery using dna microarrays phylogenetic anaylsis using parsimony (*and other methods development and application of a one-step low cost procedure to concentrate viruses from seawater samples this research was supported in part by the national institutes of health under ruth l. kirschstein national research service award ( t dk ) from the niddk and by national institutes of health grant u ai to the midwest regional center of excellence for biodefense and emerging infectious diseases research. dw holds an investigator in the pathogenesis of infectious disease award from the burroughs wellcome fund. ck is supported by an nhmrc rd wright research fellowship (id ). we would like to thank drs. gregory storch and binh-minh le for their help in the accrual and processing of the st. louis stool specimens and dr. joseph derisi and his laboratory at uc san francisco for sharing independently derived data prior to publication. the authors declare that they have no competing interests. lh performed the experiments, sequence analysis and wrote the paper. sf and gz performed sequence analysis; ck, rg and jp provided stool and sewage for analysis, dw conceived the project and helped write the paper. all authors have read and approved the final manuscript. key: cord- -nirg authors: abebe, endeshaw chekol; dejenie, tadesse asmamaw; shiferaw, mestet yibeltal; malik, tabarak title: the newly emerged covid- disease: a systemic review date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: nirg coronaviruses are large family-rna viruses that belong to the order nidovirales, family coronaviridae, subfamily coronavirinae. the novel covid- infection, caused by a beta coronavirus called sars-cov- , is a new outbreak that has been emerged in wuhan, china in december . the most common symptoms of covid- are fever, cough, and dyspnea. as per the march , , who report, more than , confirmed covid- cases and over deaths have been identified in more than countries. it is now regarded as a pandemic that seriously spread and attack the world. the primary means of transmission is person to person through droplets that occurred during coughing or sneezing, through personal contact (shaking hands), or by touching contaminated objects. so far, there is no effective therapy and vaccine available against this novel virus and therefore, only supportive care is used as the mainstay of management of patients with covid- . the mortality rate of covid- is considerable. this work aimed to provide insight on the newly emerged covid- , in the hope to gain a better understanding on the general overview, epidemiology, transmission, clinical features, diagnosis, treatment, and clinical outcomes as well as the prevention and control of covid- . coronavirus disease (covid- ) is an illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus . it was first identified amid an outbreak of respiratory illness cases in wuhan city, hubei province, china. initially, it was reported to the who on december , . on january , , the who declared the covid- outbreak a global health emergency. who declared covid- a global pandemic on march , . this work aimed to provide insight on the newly emerged covid- , in the hope to gain a better understanding on the general overview, epidemiology, transmission, clinical features, diagnosis, treatment, and clinical outcomes as well as the prevention and control of covid- . coronaviruses (cov) are a large family of pleomorphic, crown-shaped, enveloped positive sense single-stranded rna viruses that belong to the order nidovirales, family coronaviridae, subfamily coronavirinae. there are hundreds of coronaviruses, most of which are zoonotic that are present in humans and various animals (including pigs, camels, bats, and cats). they are classified into four genera rna containing viruses pathogenic for mammals, namely alpha-, beta-, gamma-, and deltacoronavirus. so far only the first two coronaviruses are identified in humans and the latter two have only been identified in animals, with bats being host to the largest genomic variety [ ] [ ] [ ] . the cov causes an illness with a wide range of clinical features that usually result in mild to moderate upperrespiratory tract illnesses, like the common cold and in some cases causing the more severe type of illness involving the respiratory, gastrointestinal, hepatic and neurologic systems. seven coronaviruses species are known to cause human disease, of which four prototypic cov cause endemic and epidemic respiratory diseases, including the alpha-coronaviruses- e and -nl , and the beta-coronaviruses oc and human coronavirus hku . due to the rapid mutation and genetic recombination of cov, three new cov have emerged since the s. these new viruses can have more serious outcomes in people, and responsible for coronavirus outbreaks for three times in the past two decades in the twenty-first century, which have emerged from animal reservoirs to cause severe disease and global concerns. those diseases are sars (severe acute respiratory syndrome) which is caused by sars-cov (beta-coronavirus), and emerged as an outbreak in late ; mers (middle east respiratory syndrome) which is caused by mers-cov (beta-coronavirus) and it is an ongoing outbreak emerged in ; and the newly emerged covid- [ ] [ ] [ ] . the novel coronavirus disease (covid- ) emerged in the huanan seafood market, where livestock animals are also traded in china on december , , and becoming a global issue to control its spread. it was initially observed in wuhan city of china as a cluster of patients with pneumonia of unknown etiology and reported to the world health organization (who) china bureau in beijing. a week later, on january , , a new coronavirus that has not been previously identified in humans, was isolated from these patients by the chinese scientists [ ] . this newly recognized strain of coronaviruses that have been found to be the etiologic agent of covid- was called severe acute respiratory syndrome coronavirus (sars-cov- ). like sars-cov and mers-cov, this novel cov is classified under the group of beta-coronavirus (fig. ) . it was initially referred to as novel coronavirus ( -ncov) but was given the official name of covid- by who on february , [ ] . similar to the sars epidemic, this outbreak has occurred during china's annual lunar new year holiday, which is the most famous traditional festival in china, during which nearly billion people travel from all sides of the country. these conditions favoured the transmission of this highly contagious disease and become severe problems in the prevention and control of the outbreak [ , ] . the current novel coronavirus disease (covid- ) that causes pneumonia is a highly infectious disease, and the ongoing outbreak has affected a huge part of populations around the world. it has been declared by who as a global public health emergency of international concern on january , [ ] . it is now spread rapidly from its first site of outbreak, wuhan city, throughout hubei province to other provinces in china and around the world. the epidemic is now reported to expand to over countries as per march , , who report. since the start of the outbreak, more than , confirmed covid- cases have been identified and over deaths have been globally reported. of these, the majority ( . %) cases were detected in china while the rest ( . %) were identified outside china. the global distribution of covid- cases as of march , who situation reports is shown in fig. . the number of countries reporting confirmed covid- cases is now increasing from time to time according to who daily report. the epidemic curve of confirmed covid- cases outside china is depicted in fig. [ ] . the total number of covid- cases is likely to be higher due to the inherent difficulties in identifying and counting mild and asymptomatic cases, especially in those developing countries having poor settings for case detection. besides, the still-insufficient testing capacity for covid- , which means many suspected and clinically diagnosed cases are not yet counted in the [ ] denominator [ ] . furthermore, covid- infected more people than either of its two predecessors, sars and mers. sars which was emerged and declared by who as an outbreak in november in guangdong province of china and contained on july , , with a total of sars cases and deaths across countries while mers emerged in in saudi arabia and is still ongoing outbreak and is thus far responsible for confirmed cases and deaths across countries [ , ] . the novel covid- more commonly affected males, and the median age range of patients is to years. nearly all reported cases have occurred in adults (median age years). generally, older people are twice as likely to have serious covid- illness. just over % of cases were under years of age, of which fewer than % developed severe or critical diseases [ , , ] .. based on the study done on , cases in china by wu and mcgoogan, the overall case-fatality rate (cfr) of covid- was . %. the cfr was higher in those cases aged years and above ( . %) and among critical covid- cases ( . %). the cfr was also higher among those with preexisting comorbid conditions: . % for cardiovascular disease, . % for diabetes, . % for chronic respiratory disease, . % for hypertension, and . % for cancer [ ] . despite much higher cfrs for sars ( . %) and mers ( . %), covid- has led to more total deaths due to the large number of cases [ ] . research evidences suggest sars-cov- , like sars-cov and mers-cov, is zoonotic that was transmitted to from animals. it has been declared that sars-cov- is originated from wild bats. however, a considerable transmission of covid- is occurring among people who are in close contact with one another (within about ft) and also through droplets produced when an infected person coughs or sneezes. these droplets can be inhaled into the lungs or drops into the nearby mouths or noses of people [ ] . this human-to-human transmission of covid- is confirmed by a recent report from many infected healthcare workers in wuhan. in addition, it may be possible that a person can get covid- by touching the virus-infected inanimate objects and having contact with their own mouth, nose, or possibly their eyes unwashed, but this is not thought to be the main way the virus spreads. the most symptomatic (the sickest) the people are, the most contagious they thought to be. but some spread might be possible with this new coronavirus during the asymptomatic phase though this is not thought to be the main way the virus spreads [ , ] . the transmission also occurs in hospital settings though it is not the major means of covid spread. this is supported from the study by wang and his colleagues in which covid- affected health professionals, ( %) and hospitalized patients, ( . %) and hospitalacquired transmission was suggested as the presumed mechanism of infection [ ] . hospital-acquired infection occurred in . % of patients as per a report of yang et al [ ] . moreover, the virus that causes covid- seems to show a "community spread" in which the virus is spreading easily and sustainably in the community and the people have been infected without noticing how or where they contacted with the infection and scientists are still trying to determine how sars-cov- spread to people [ , ] . as per the findings from a small-scale study by chen and his colleagues it has been reported that covid- is not transmitted vertically from infected pregnant women to their fetus. however, this needs further investigation to surely arrive at any conclusion. the study also reported that there is currently no evidence for intrauterine infection caused by vertical transmission in women who develop covid- pneumonia in late pregnancy [ ] . the epidemic curves reflect as it may be a mixed outbreak pattern, with early cases suggestive of a continuous common source, potentially zoonotic spillover at huanan seafood wholesale market, and later cases suggestive of a propagated source as the virus began to be transmitted from person to person. thus, covid- rapidly spread from a single city to the entire country of china in just days, which quickly overwhelmed the health and public health services of china, particularly in wuhan city and hubei province [ ] . several factors have been forwarded for the rapid spread of this new virus: one is wuhan is the capital of china's hubei province, with more than million inhabitants, and it is a major hub of transportation, which increases person-toperson contact and increases the chance of exporting cases to other locations. the unavailability of effective antiviral therapy and vaccine makes the disease difficult to prevent and control the spread. as a result, the outbreak spread to many chinese provinces, and then to the asian continent. the first non-chinese case of the infection was reported from thailand on january , . the source of infection was from a chinese tourist who has traveled to thailand. other cases are then continued to be reported in many countries of the world [ , , ] . more than % of covid- illnesses are asymptomatic or can be symptomatic with mild symptoms such as respiratory symptoms, fever, cough, shortness of breath, and breathing difficulties. in some patients, covid- results in severe illness with serious complications: severe pneumonia, acute respiratory distress syndrome (ards), acute respiratory failure, pulmonary edema, sepsis, septic shock or multiple organ failure, and even death [ ] . symptoms may appear - days after exposure. the median duration from onset of symptoms to radiological confirmation of pneumonia was days while the median duration from onset of symptoms to icu admission was . days [ ] . the most common and frequently reported symptoms of covid- are fever ( %), cough ( %), and dyspnea ( . %) whereas less common symptoms are myalgia, malaise, arthralgia, chest pain, nasal congestion, runny nose, headache, sore throat, and diarrhea (fig. ) [ , , ] . but not all these symptoms are present in covid- cases. for instance, among critically ill patients, six ( %) did not experienced fever until - days after the onset of symptoms related to sars-cov- infection [ ] . covid- infected pregnant women have been reported to have a similar pattern of clinical characteristics as the nonpregnant adult patients [ ] . clinical diagnosis of covid- can be made based on symptoms, exposures, and chest imaging. even though covid- manifest with different symptoms, none of these symptoms are present in every patient and there are no specific signs or symptoms that could suggest covid- compared to symptoms and signs of respiratory illnesses caused by other viruses, such as influenza and common cold [ , ] . therefore, a thorough and meticulous clinical examination needs to be done before arriving at the final diagnosis of covid- (table ) . besides, a covid- suspected individual needs to be evaluated and confirmed for sars-cov- using viral nucleic acid test called quantitative real time-pcr (qrt-pcr) using respiratory tract samples (e.g, throat swabs). samples from amniotic fluid, cord blood, and neonatal throat swab can also be used for the diagnosis of sars-cov- using pcr in pregnant women [ ] . moreover, chest ct might have a high diagnostic value because of its typical images of virus infection, high accuracy with a low false-negative rate, and time efficiency. therefore, in addition to using qrt-pcr which is the gold standard for the diagnosis of covid- pneumonia, it is beneficial to do relevant clinical examinations including complete blood counts (cbc), inflammatory markers and chest radiography and a comprehensive evaluation of a patient's medical history, epidemiological exposure, and symptoms [ , ] . from the cbc, lymphopenia ( %) appears as a prominent and the most likely occurring laboratory finding along with leukopenia ( - %), leukocytosis ( - %), and thrombocytopenia ( %). the increased concentrations of transaminases (alt or ast) are also the laboratory findings of covid- , in which elevated transaminases were noted in % of whom extreme elevations are rare. inflammatory markers such as crp and esr are elevated ( and % respectively) while procalcitonin is normal in most cases. chest x-ray and ct findings show bilateral infiltrates and unilateral involvement in and %, respectively [ , , ] . for easy reference and surveillance, case definitions of covid- are set by who based on the currently available informations [ ] and defines covid- case as: i. if a patient with a severe acute respiratory infection (fever, cough, and requiring admission to hospital), and with no other etiology that fully explains the clinical presentation and a history of travel to or residence in a country/area or territory reporting local transmission during the days prior to symptom onset, or ii. a patient with an acute respiratory illness and having been in contact with a confirmed or probable covid- case in the last days prior to the onset of symptoms or iii. a patient with a severe acute respiratory infection (fever and at least one sign/symptom of respiratory disease (e.g., cough, shortness breath) and requiring hospitalization and with no other etiology that fully explains the clinical presentation. if a suspect case for whom testing for covid- is inconclusive. inconclusive being the result of the test reported by the laboratory. or a suspect case for whom testing could not be performed for any reason. if a person with laboratory confirmation of -ncov infection, irrespective of clinical signs and symptoms. patient supportive care is typically the mainstay of treatment of covid- as there are no specific effective antiviral therapies that have been identified so far. standard supportive management including care to relieve symptoms and advanced vital organ support are indicated for respiratory disease and complications [ , ] . mechanical ventilation is the main supportive treatment for critically ill patients, which involves invasive methods such as endotracheal intubation and noninvasive method like supplementary oxygen therapy (target spo ≥ % in nonpregnant adults and spo ≥ - % in pregnant patients) [ ] . in addition, other supportive cares that should be given for severely ill patients involve close monitoring for signs of deterioration, conservative fluid management, giving antipyretics and /or analgesics, administering appropriate empirical therapy immediately (within an hour) of sepsis identification, and managing sepsis and septic shock by following sepsis guideline [ ] . without sufficient evidence, patients were given intravenous glucocorticoids and antiviral agents. intravenous glucocorticoids were commonly used in patients with table symptoms of covid- compared to influenza and common cold [ , ] . sars and mers pneumonia though their efficacy remains controversial and their use to treat covid- infection is also controversial. hence, systemic corticosteroids should not be routinely given for the treatment of viral pneumonia or ards outside of clinical trials unless they are indicated for another reason: septic shock or other disease processes. currently, in the usa, remdesivir, which was tested against ebola, and kaletra (lopinavir and ritonavir combination) are suggested to be the potential treatments against the novel coronavirus. favorable outcome was brought with the intravenous remdesivir treatment of the first covid- case in the usa. but clinical trials are underway to come up with solid evidence to recommend these drugs to routinely prescribe for covid- and thus, the ongoing clinical trials might shed some light on the safety and efficacy of these drugs as treatment [ , , ] . at this time, it is difficult to predict the mortality of covid- as it has been fluctuating due to the source of information. but the who estimated the overall cfr between and % [ ] . however, the mortality of critically ill patients with covid- pneumonia is considerable. according to a single-centered, retrospective study by yang and his colleagues, the survival time of the nonsurvivors is likely to be within - weeks after icu admission. for instance, of those critical patients requiring mechanical ventilation, % had died by days. moreover, the poor clinical outcome of covid- patients was reported to be associated with older age and underlying comorbidities. older patients (> years) with comorbidities and ards are at increased risk of death [ ] . the high mutation rate, different means of transmission of the novel virus as well as unavailability of the vaccine against covid- makes it difficult to control the spread. developing a vaccine is underway and hopes to begin a phase trial within months. as a consequence, countries are now focusing on traditional public health outbreak response tactics such as isolation, quarantine, social distancing, and community containment as targeted antiviral drugs and vaccines are not yet available for covid- [ , ] . the best way to prevent illness is to promptly implement infection control measures to avoid being exposed to this virus. infection control measures are integral parts of the clinical management of patients and should be initiated at the point of patient's admission to the hospital. standard precautions should always be routinely applied in all areas of health care facilities. the who advised the world the shift from containment to mitigation would be wrong and dangerous as covid- is a controllable pandemic. so, we can control or contain the person-to-person spread of covid- infection by doing the following standard precautions/recommendations [ , ] . regular hand-washing often with soap and water for at least s, especially after going to the bathroom; before eating; and after blowing your nose, coughing, or sneezing. if soap and water are not available, use an alcohol-based hand sanitizer containing at least % alcohol. always wash hands with soap and water if hands are visibly dirty. avoid touching your eyes, nose, or mouth with unwashed hands. avoid close contact with anyone showing symptoms of respiratory illness such as coughing and sneezing. stay home when you are sick. covering mouth and nose with a tissue when coughing and sneezing, then throw the tissue in the trash. thoroughly cooking meat and eggs. clean and disinfect frequently touched equipment, objects, and surfaces using a regular household cleaning spray or wipe. prevention of needle-stick or sharps injury safe waste management and environmental cleaning use of personnel protective equipment (ppe) to avoid direct contact with patients' blood, body fluids, secretions (including respiratory secretions), and non-intact skin. following center for disease control (cdc) recommendation for facemask: cdc recommends facemasks to those people who show symptoms of covid- to help prevent the spread of the disease to others. otherwise, cdc does not recommend that healthy people wear a facemask to protect themselves from covid- . people who think they may have been exposed to covid- should contact their healthcare provider immediately to be treated immediately and to prevent the spread [ , ] . in conclusion, the novel covid- infection is a new outbreak that has been emerged in wuhan, china in december . it is caused by a novel beta-coronavirus, resulting from genetic recombination, called sars-cov- , the most common symptoms of covid- are fever, cough, and dyspnea. as per the march , who report, more than , confirmed covid- cases and over deaths have been identified in countries. it is now regarded as a pandemic that seriously spread and attack the world. the primary means of transmission is person to person through droplets that occurred during coughing or sneezing, through personal contact (shaking hands), or by touching contaminated objects. the overall cfr is between and %. this figure is however higher in the elderly and those with underlying health conditions. older patients (> years) with comorbidities and ards are at increased risk of death. so far, there is no effective therapy and vaccine against this novel virus and therefore, only supportive care is used as the mainstay of clinical management of patients with covid- . infection prevention and control measures are integral parts of the clinical management and are the best option we currently have for containment of covid- spread. coronavirus genomics and bioinformatics analysis viruses bats: important reservoir hosts of emerging viruses host factors in coronavirus replication sars and other coronaviruses as causes of pneumonia coronavirus as a possible cause of severe acute respiratory syndrome isolation of rhinoviruses and coronaviruses from colds in adults epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study covid- three emerging coronaviruses in two decades: the story of sars, mers, and now covid- characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention clinical characteristics and intrauterine vertical transmission potential of covid- infection in nine pregnant women: a retrospective review of medical records novel coronavirus ( -ncov) situation reports -novel coronavirus ( -ncov): estimating the case fatality rate: a word of caution clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? international journal of epidemiology from sars-cov to wuhan -ncov outbreak: similarity of early epidemic and prediction of future trends the continuing -ncov epidemic threat of novel coronaviruses to global health -the latest novel coronavirus outbreak in wuhan, china transmission of -ncov infection from an asymptomatic contact in germany clinical characteristics of hospitalized patientswith novel coronavirusinfected pneumonia in wuhan clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study novel coronavirus . . surveillances: ncperetv. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- )-china. china cdc weekly interim clinical guidance for management of patients with confirmed novel coronavirus ( -ncov) infection first case of novel coronavirus in the united states risk for transportation of novel coronavirus disease from wuhan to other cities in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. the first draft was designed by endeshaw chekol abebe and all the authors contributed equally in compilation and editing and finalizing the review. the author read and approved the final manuscript. not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors have no competing interests. key: cord- -xrit w i authors: feng, bo; zhao, lihong; wang, wei; wang, jianfang; wang, hongyan; duan, huiqin; zhang, jianjun; qiao, jian title: investigation of antiviral state mediated by interferon-inducible transmembrane protein induced by h n virus and inactivated viral particle in human endothelial cells date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: xrit w i background: endothelial cells are believed to play an important role in response to virus infection. our previous microarray analysis showed that h n virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein (ifitm ) in human umbilical vein endothelial cells (huvecs). in present study, we deeply investigated the expression patterns of ifitm and ifitm -mediated antiviral response induced by h n virus infection and inactivated viral particle inoculation in huvecs. epithelial cells that are considered target cells of the influenza virus were selected as a reference control. methods: first, we quantified the expression levels of ifitm in huvecs induced by h n virus infection or viral particle inoculation using quantitative real-time pcr and western blot. second, we observed whether hemagglutinin or neuraminidase affected ifitm expression in huvecs. finally, we investigated the effect of induced-ifitm on the antiviral state in huvecs by sirna and activation plasmid transfection. results: both h n virus infection and viral particle inoculation increased the expression of ifitm without elevating the levels of interferon-ɑ/β in huvecs. ha or na protein binding alone is not sufficient to increase the levels of ifitm and interferon-ɑ/β in huvecs. ifitm induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of huvecs. conclusions: our results showed that inactivated viral particle inoculation increased the expression of ifitm at mrna and protein levels. moreover, the induction of ifitm expression mediated the antiviral state in huvecs. h n influenza virus exists on all continents except antarctica and is the most common subtype of influenza viruses isolated from poultry (chickens and ducks) in china [ , ] . h n viruses have been isolated from pigs and humans with influenza-like illness in hong kong and mainland china [ ] [ ] [ ] [ ] [ ] [ ] [ ] , demonstrating that the h n influenza virus could cross species barriers and expand its host range from birds to mammalians. recently, a research showed that h n viruses and pandemic h n viruses have high genetic compatibility and they can produce higher pathogenic reassortment in experimental condition [ ] . in addition, h n viruses provide their six inner genes to contribute to the evolution of the h n , h n and h n viruses that cause severe human respiratory infections in china [ ] [ ] [ ] . all these features indicate that h n virus has a considerable public health threat. thus, it is valuable to reveal the pathogenesis of h n influenza virus infection and the innate immune responses of host to the h n viruses. pathogen invasion could be recognized by pattern recognition receptors (prrs) and results in production of interferon [ ] . the type i interferon binds to interferon-α/β receptors and activates jak-stat signaling pathway, resulting in the expression of hundreds of interferonstimulated genes (isgs) [ ] . products of these genes are mostly antiviral proteins which are essential for mediating the antiviral state of the host. interferon-induced transmembrane proteins (ifitms) were identified nearly years ago, and although other functions have been proposed, the primary role of ifitm proteins seems to be antiviral. ifitm proteins are a family of small transmembrane proteins that are induced strongly by interferon, but that are also expressed basally in several cell types and lines [ , ] . to date, three ifitm proteins (ifitm , ifitm and ifitm ) have been identified to display broad spectrum antiviral activity in human and mice [ , ] . especially, the ifitm and ifitm were proven to be more effective in resisting influenza virus infection [ , ] . the sirna-mediated depletion of ifitms significantly increased h n virus titers in primary lung fibroblasts and in hela cells, while overexpression of ifitm , , could improve resistance to the h n influenza in a and mdck cell lines [ ] . although the antiviral activity of ifitms has been identified in multiple types of cells, the antiviral activity of ifitm has not been reported in endothelial cells infected with h n virus or inoculated with inactivated viral particle. generally, airway epithelial cells are considered to be the main target cells of influenza viruses because of expressing two kinds of influenza virus receptors (α- , and α- , -linked sialic acid receptors) [ ] [ ] [ ] . but the more and more evidences indicated that endothelial cells might play an important role in response to influenza virus infection. most endothelial cells are also showed to express two kinds of influenza virus receptors which provide potential possibility for influenza virus infection in endothelial cells [ ] . h n and h n influenza viruses have been proven to directly infect human lung microvascular endothelial cells and replicate in endothelial cell lines [ , ] . several studies indicated that endothelial cells might be the source of the cytokines and involved in lung injury during influenza virus infection [ ] [ ] [ ] [ ] . so, it is important to understand the cellular responses in endothelial cell during influenza virus infection. our previous study revealed that h n virus could infect human umbilical vein endothelial cells (huvecs) and induce high level expression of several isgs, especially ifitm [ ] . the microarray results showed that both h n virus infection and inactivated viral particle inoculation increase the expression of ifitm at transcription level, and the viral particle inoculation induces a higher level ( . folds) of ifitm than that ( . folds) induced by virus infection. however, the level of ifitm needs to be quantified by real-time pcr and western blot, and little is known about the antiviral activity of ifitm induced by h n virus and viral particle in huvecs. the present study aimed to quantify the expression of ifitm induced by h n virus and viral particle and investigate the antiviral state mediated by ifitm in huvecs. and the results showed that both h n virus infection and viral particle inoculation significantly increased the expression of ifitm at mrna and protein levels, and the ifitm protein induced by viral particle inoculation significantly enhanced the antiviral state of huvecs against h n virus infection. we first quantified the expression levels of ifitm in huvecs induced by h n virus infection or viral particle inoculation using quantitative real-time pcr and western blot. secondly, we detected levels of interferonɑ/β using elisa kits. thirdly, we observed the effect of ha and na on ifitm expression, and located the position of inactivated viral particles in huvecs. finally, we investigated ifitm -mediated antiviral response by sirna and activation plasmid transfection. to compare the expression patterns of ifitm between endothelial cells and epithelial cells, all of the above experiments were performed on human epithelial cells at the same time. human umbilical vein endothelial cells (huvecs, crl- , atcc), human bronchus epithelial cells (beas- bs, crl- , atcc) were used for detecting the expression of ifitm and secretion of interferon-ɑ/β. madin darby canine kidney cells (mdck, ccl- , atcc) were used for plaque assay. these cell lines were cultured in dmem (gibco) supplemented with % fbs (gibco), u/ml of penicillin g and μg/ml of streptomycin (gibco) at °c in a % co incubator. the cells in passages ~ were seeded into six-well plates and cultured for h before each experiment. the h n virus used in this study was a/chicken/hebei/ / (h n ) (ck/hb/ / ). the complete genome sequences of the virus are available from genbank under accession numbers fj -fj . the viruses were propagated in -day-old embryonated eggs from specificpathogen-free (spf) hens at °c for to h. virus titers were determined by plaque assay. our previous study demonstrated that exogenous trypsin was required for the efficient replication of the h n virus in huvecs [ ] . thus, . μg/ml exogenousl- -tosylamide- -phenylethyl chloromethyl ketone (tpck)-treated trypsin was added to the medium for all experiments. to inspect the interaction between viral particles and huvecs, we created inactivated h n viral particles by using . % β-propionolactone (serva electrophoresis) according to previous description [ ] , and plaque assay was used to evaluate whether viral replicative capacity was completely destroyed. to quantify the levels of ifitm induced by h n virus and viral particle, huvecs or beas- bs were divided into three groups: control group (inoculation with virusfree media), h n virus group (infection with virus at a multiplicity of infection (moi) of ) and viral particle group (inoculation with viral particles at a moi of ). to detect the effect of ha and na (sino biological) on the mrna expression of ifitm , huvecs or beas- bs were divided into control group, low concentration group, high concentration group. cells in control group were inoculated with free media, cells in low and high concentration groups were respectively treated with . μg/ml and μg/ml ha or na [ , ] . rna in each group used for real-time pcr analysis was extracted using trizol reagent (invitrogen) at , and h after treatment. cdna was synthesized using random hexamer and a superscript iii reverse transcriptase kit (invitrogen). real-time pcr was performed on the cdna for rna quantitation using ex taqmix (takara bio) and eva green (biotium) for ifitm . gapdh was amplified in parallel with the target genes as an endogenous control and all samples were analyzed in triplicate. the fold changes of specific mrna from different groups compared to control group. the primer sequences were used as follows: gapdh, f ( ′ to ′): acaacttt ggtatcgtggaaggac and r ( ′ to ′): agggatgatgttctggagagcc, ifitm , f ( ′ to ′): actgaaacgacaggggaaag and r ( ′ to ′): gaacagggaccagacgacat. cells in each group used for western blot analysis were collected at , h after treatment and lysed with ripa lysis buffer (cell signaling technology). the cell lysates were centrifuged and the resultant supernatants were resolved by sds-polyacrylamide gel electrophoresis (sds-page) and then transferred onto polyvinylidene difluoride (pvdf) membranes (roche). the membranes were blocked with % skim milk and probed with a monoclonal antibody to β-actin (santa cruz biotechnology), ifitm (sigma-aldrich). after a further incubation with peroxidase (hrp) conjugated secondary antibody (origene). proteins were visualized using enhanced chemiluminescence. the relative protein level of ifitm to β-actin was analyzed by image j software. elisa assay was used to quantify the levels of interferonɑ/β. supernatant in each group was collected at , and h postinfection, and analyzed by elisa kits (r & d systems). according to previous description [ , ] , cells in positive control group were treated with poly i: c (sigma) at the concentration of μg/ml. the plaque assay was used to detect the virus titers propagated in eggs and cell culture supernatant. plaque assay was performed on mdck cells as described previously [ ] . briefly, mdck cells were seeded into -well plates. confluent monolayers were washed with phosphate buffered saline (pbs) and infected with h n virus or cell culture supernatants. the inoculum was discarded after incubation for h and the remaining cells were washed with pbs. an overlay consisting of a mixture of . % agarose (lonza) and doublestrength dmem with . μg/ml tpck-trypsin (worthington) was added to the above cells and incubated at °c for h. plaques were stained with . % crystal violet and counted. for interference assay, cells were transfected with control sirna or ifitm specific sirna using lipofectamine transfection reagent kit (invitrogen). ifitm specific and control sirna used in the study were purchased from santa cruz biotechnology, and the sirna for human cells is a pool of target-specific - nt sirnas designed to knock down gene expression. according to instructions of products, we firstly prepared mixtures ( . μm sirna and . μl lipofectamine ) of sirna and lipofectamine transfection reagents using opti-mem media. to interfere the expression of ifitm , cells were infected with h n virus or viral particle and incubated for h, then cells were covered with mixtures for h at °c in a % co incubator. the interference effect of ifitm specific sirna was detected by western blot at h postinfection. for overexpression assay, cells were transfected with control plasmid or ifitm crispr activation plasmid (santa cruz biotechnology) using lipofectamine transfection reagent kit (invitrogen). the ifitm crisper activating plasmid is a synergy activation medium (sam) transcriptional activation system designed to specifically upregulate gene expression. according to instructions of products, we firstly prepared mixtures ( μg plasmid and . μl lipofectamine ) of plasmid and lipofectamine transfection reagents using opti-mem media. then cells were covered with the mixtures for h at °c in a % co incubator. the overexpression level of ifitm was detected by western blot at h after transfection. immunofluorescence was used to locate the position of viral particles in cells. according to previous description [ ] , huvecs or beas- bs were directly fixed in % paraformaldehyde, permeabilized with . % triton x- . after blocking with phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) for h at °c. cells were labeled with anti-nucleoprotein (np) antibody a- (aviva systems biology) overnight, followed by stained with fitc conjugated anti-rabbit secondary antibody (origene) for h at °c. the images were captured using olympus fluorescence microscopy. the data were expressed as means ± standard deviations (sd). all the statistical tests were performed using graphpad prism software (version . ). statistical significance of differences were determined using the student's t-test or one-way analysis of variance (anova). p < . was considered statistically significant. according to our previous microarray results, both h n virus infection and inactivated viral particle inoculation upregulate the expression of ifitm at transcriptional level. here we used real-time pcr and western blot to quantify the levels of ifitm induced by virus infection or viral particle inoculation in huvecs and beas- bs. results showed that both h n virus infection and viral particle inoculation increased the expression of ifitm at mrna and protein levels in huvecs and beas- bs (fig. ). compared to virus group, viral particle inoculation induced higher levels of ifitm at the mrna level at h and at protein level at h (p < . , anova) in huvecs (fig. a-c) . in contrast, h n virus infection induced higher expression of ifitm at mrna level at h, h, h (p < . , anova), and higher levels of ifitm protein at h, h (p < . , anova) in beas- bs ( fig. d-f ). our data showed that h n virus infection and viral particle inoculation induced different kinetics of ifitm expression in/ between huvecs and beas- bs. according to previously described, expression of ifitm proteins are potently induced by type i interferon [ ] . to determine whether interferon-α/β were involved in the expression of ifitm induced by h n virus and viral particle, we treated huvecs and beas- bs with h n virus or viral particle at a moi of and incubated for h. supernatant in each group was collected at , and h postinfection for elisa assay. results showed that there was no significant difference (p > . , anova) between treated and untreated control group at each time point in huvecs (fig. a, b) , suggesting that interferon-α/β did not participate in the induction of ifitm in huvecs. however, different results were observed in beas- bs. compare to control group, levels of interferon-α/β were significantly upregulated at , and h postinfection (p < . , anova), viral particle inoculation just induced a higher level of interferon-α (p < . , anova) at h (fig. c, d) . in addition, h n virus infection also induced higher levels of interferonα/β than that induced by viral particle inoculation in beas- bs (fig. c, d) . the results demonstrated that h n virus and viral particle might share different mechanisms in the induction of ifitm expression in huvecs and beas- bs. ha and na proteins had no effect on the expression of ifitm figs. , showed that h n virus infection and viral particle inoculation increased the expression of ifitm independently of interferon-α/β in huvecs. thus, we investigated whether envelope proteins were involved in the induction of ifitm expression. we incubated huvecs with ha or na protein at different concentrations. supernatant in each group was collected at , and h for elisa assay, cells were extracted for real-time pcr analysis. the results showed that interferon-α/β levels were not significantly increased after treatment with ha or na (p > . , anova) in both beas- bs and huvecs (fig. ) . as shown in fig. , the mrna levels of ifitm were not significantly increased at , and h (p > . , anova) in huvecs and beas- bs. the results suggested that the ha or na protein binding alone is not sufficient to induce the expression of ifitm in huvecs or beas- bs. location of viral particle viral particle inoculation significantly increased the expression of ifitm independently of interferon-α/β in huvecs, and the binding of ha or na protein alone is not sufficient to increase the ifitm level. we suspected that effect of viral particle on ifitm expression was generated inside the cells. then we located viral particles using immunofluorescence. cells were inoculated with viral particles at a moi of and incubated for h. according to previous description [ , ] , viral particles were labeled with anti-np antibody a- and fitc conjugated secondary antibody at h after inoculation. results showed that np-positive cells were observed after inoculation with viral particles (fig. ) . the results indicated that the cellular interaction between intracellular molecules and viral particles might be involved in the induction of ifitm expression in huvecs. to determine the antiviral activity of ifitm protein induced by h n virus infection, huvecs or beas- bs were infected with h n virus at moi of and incubated for h, then cells were transfected with ifitm specific sirna or control sirna for h. as shown in fig. a , ifitm specific sirna transfection successfully knocked down the expression of ifitm induced by h n virus infection in huvecs. however, the virus titers were just increased by . ± . % (p > . , t-test) compared with the virus fig. quantitation of ifitm induced by h n virus or inactivated viral particle in huvecs and beas- bs. huvecs and beas- bs were treated with h n virus (i.e., virus) or viral particle (i.e., particle) at moi of . cells used for rt-pcr and western blot analysis were collected at different time points. a the expression of ifitm at mrna level in huvecs. b, c the expression of ifitm at protein level in huvecs, and the relative protein level to β-actin. d the expression of ifitm at mrna level in beas- bs. e, f the expression of ifitm at protein level in beas- bs, and the relative protein level to β-actin. * means particle group and virus group compared with control group (*, p < . . **, p < . , anova). # means particle group compared with h n virus group (#, p < . . ##, p < . , anova) fig. production of interferon-α/β by huvecs and beas- bs inoculated with h n virus or viral particle. huvecs and beas- bs were inoculated with h n virus or viral particle at a moi of , and then supernatants were collected at , , h. levels of interferon-α/β were detected using elisa kits. values represent the means from three independent experiments plus standard deviations. a, b the concentration of interferon-α/β in culture supernatants of huvecs. c, d the concentration of interferon-α/β in culture supernatants of beas- bs. *means particle group and virus group compared with control group (*, p < . . **, p < . , anova). # means particle group compared with h n virus group (#, p < . . ##, p < . , anova) fig. evaluation of interferon-α/β in huvecs and beas- b incubated with hemagglutinin (ha) and neuraminidase (na). huvecs and beas- bs were incubated with ha or na at concentrations of . , μg/ml, supernatants were collected at , , h. levels of interferon-α/β were determined using elisa kits. values represent the means from three independent experiments plus standard deviations. a, b the concentration of interferon-α/β in culture supernatants of huvecs. c, d the concentration of interferon-α/β in culture supernatants of beas- bs group (control sirna) (fig. c) . similarly, the expression of ifitm induced by h n virus infection was also successfully interfered by ifitm specific sirna transfection in beas- bs (fig. b) . and the virus titers were just increased by . ± . % (p > . , t-test) compared with virus group (control sirna) (fig. d) . the results suggested that ifitm induced by h n virus infection did not mediate the antiviral response in huvecs and beas- bs. huvecs or beas- bs were inoculated with viral particles at moi of and incubated for h, then cells were transfected with sirna for h before infected with h n virus. results showed that sirna transfection knocked down the expression of ifitm induced by viral particle inoculation in huvecs and beas- bs huvecs and beas- bs were infected with h n virus at moi of and incubated for h, then cells were transfected with control sirna or ifitm specific sirna for h. virus titer in each group was detected using plaque assay at h postinfection. the effect of ifimt specific sirna on ifitm expression was detected using western blot. a ifitm protein level after transfected with sirna in huvecs. b ifitm protein level after transfected with sirna in beas- bs. c virus titers in huvecs transfected with control sirna or ifitm specific sirna. compared to virus group (control sirna), the virus titer in virus + sirna group (ifitm specific sirna) was increased by . ± . % (p > . , t-test). d virus titers in beas- bs transfected with control sirna or ifitm specific sirna. compared to virus group (control sirna), the virus titer in virus + sirna group (specific sirna) was increased by . % ± . % (p > . , t-test) fig. antiviral activity of ifitm induced by viral particle inoculation in huvecs and beas- bs. huvecs and beas- bs were inoculated with viral particle at moi of and incubated for h, then cells were transfected with control sirna or ifitm specific sirna for h before infected with h n virus at moi of . virus titer of each group was detected by plaque assay at h postinfection. the effect of ifimt specific sirna on ifitm expression was detected using western blot. a ifitm protein level after transfected with sirna in huvecs. b ifitm protein level after transfected with sirna in beas- bs. c virus titers in huvecs transfected with control sirna or ifitm specific sirna. compared to particle group (control sirna), the virus titer in particle + sirna group (ifitm specific sirna) was increased by . ± . % (p < . , t-test). d virus titers in beas- bs transfected with control sirna or ifitm specific sirna. compared to particle group (control sirna), the virus titer in particle + sirna group (specific sirna) was increased by . ± . % (p > . , t-test). * means particle group compared with particle + sirna group (*, p < . , t-test) (fig. a, b) . compared to particle group, the virus titers were significantly increased by . ± . % (p < . , ttest) after transfection with ifitm specific sirna (fig. c) . in contrast, the virus titers were only increased by . ± . % (p > . , t-test) after transfection with ifitm specific sirna in beas- bs (fig. d) . the results indicated that the ifitm protein induced by viral particle inoculation significantly enhanced the antiviral state of huvecs. huvecs or beas- bs were transfected with plasmid for h before infected with h n virus. the results showed that transfection with ifitm crispr activation plasmid upregulated the expression of ifitm in huvecs and beas- bs (fig. a, b) . compared to control group, the virus titers were significantly decreased by . ± . % (p < . , t-test) in beas- bs transfected with ifitm crispr activation plasmid (fig. c) , the virus titers were significantly decreased by . ± . % (p < . , t-test) in huvecs transfected with ifitm crispr activation plasmid (fig. d ). the expression of isgs is an early response of host to virus infection, and their products confers host the antiviral state which inhibits the entry process or replication of invading virus. to date, multiple proteins translated by isgs have been validated as antiviral proteins, such as protein kinase r (pkr), myxovirus-resistance proteins, isg , schlafen and so on. ifitm proteins are recently identified antiviral factors that play critical roles in the intrinsic and interferon-mediated control of virus infection [ , ] . since initially identified by a sirna screen for factors that restrict influenza virus replication, more and more researches revealed the inhibition effect of ifitms on enveloped viruses through affecting the interaction between virus envelope proteins and endosomal or lysosomal [ ] . recently, a study demonstrated that depletion of ifitm with sirna increased titers of h n virus in primary lung fibroblast cells and in hela cell line, overexpression of ifitm resisted h n virus infection in a and mdck cell lines [ ] . in this study, our results showed that overexpression of ifitm significantly reduced the virus titers in huvecs and beas- bs (fig. ) . the present data reaffirmed the restriction effect of ifitm on influenza virus infection. to investigate whether ifitm induced by h n virus infection and viral particle inoculation mediated the antiviral response in huvecs, we detected the virus titers in huvecs transfected with ifitm specific sirna or control sirna. the results showed that ifitm induced by h n virus infection could not enhanced the antiviral state in huvecs. in contrast, ifitm induced by viral particle inoculation significantly enhance the fig. overexpression of ifitm significantly reduced virus titers in huvecs and beas- bs. huvecs and beas- bs were transfected with control plasmid (control) or ifitm crispr activation plasmid (plasmid) for h, then cells were infected with h n virus at moi of . virus titer of each group was detected by plaque assay at h postinfection. the overexpression of ifimt was detected by western blot. a ifitm protein level after transfected with plasmid in beas- bs. b ifitm protein level after transfected with plasmid in huvecs. c virus titers in beas- bs transfected with control plasmid or ifitm crispr activation plasmid. compared to control group (control plasmid), the virus titer in plasmid group (ifitm crispr activation plasmid) was decreased by . . ± . % (p < . , t-test). d virus titers in huvecs transfected with control plasmid or ifitm crispr activation plasmid. compared to control group (control plasmid), the virus titer in plasmid group (ifitm crispr activation plasmid) was decreased by . ± . % (p < . , t-test). * means control group compared with plasmid group (**, p < . , t-test) antiviral response in huvecs (fig. ) . taking into account the above results, we hypothesized that virus replication preceded the expression of ifitm in initially infected cells. a previous study demonstrated that ifitm efficiently restricted influenza virus and ifitm modestly restricted influenza virus, and the expression patterns of ifitms are likely to be an independent determinant of viral tropism [ ] . our previous microarray results showed that h n virus and viral particle do not induce the expression of ifitm in huvecs [ ] . we speculated that this might be a unique response of huvecs to h n virus infection or viral particle inoculation. taken together, our data may offer further insight into the innate immune response of endothelial cells to influenza virus infection. in the present study, the level of ifitm induced by viral particle inoculation was higher than that induced by h n virus infection in huvecs (fig. ) . the observation differed to what we generally expected, so it is worthy of thinking the sense of ifitm expression upregulated by viral particle. moreover, a recent study indicated that h n influenza virus infection induces the expression of ifitm in lung, heart and liver in balb/c mice [ ] . in vivo, the basal lamina with an average thickness of μm is the only structure which separates epithelial cells and endothelial cells [ ] . death of infected epithelial cells creates gaps and the released virus particles readily access to endothelial cells [ , ] . thus, the released virus particles may stimulate the antiviral response in endothelial cells. in addition, cytokines produced by epithelial cells could further activate neighboring endothelial cells during influenza virus infection [ ] . so it is conceivable that interferon-α/β released by infected epithelial cells could readily induce the expression of ifitm proteins in endothelial cells. taken together, we may consider to enhance the antiviral state of host by stimulating the respiratory tract with inactivated viral particles. previous studies indicated that h n virus infection up-regulate the expression of type i interferon in human pulmonary microvascular endothelial cells and induce high levels of interferon-β in huvecs [ , ] . however, our results showed that expression of ifitm induced by h n virus and viral particle independently of interferon-α/β in huvecs (fig. ) . although a recent study demonstrated that human ifitm and mouse ifitm are induced by cytokines of the gp family (such as il- ) [ ] , we failed to find a possible mechanism involved in ifitm expression. thus, we investigated the effects of ha and na proteins on the expression of ifitm in huvecs. the results showed that treatment with ha or na could not upregulate the levels of interferon-α/β and ifitm (figs. , ) . virus rna activates the cellular antiviral response has been widely reported. in the present study, we did not investigate the effect of rna of h n virus on the antiviral response in endothelial cells. however, our results showed that h n virus could infect and replicate in huvecs with invalid effect on antiviral response of huvecs. the results indicated that rna of h n virus may play an insignificant role in the expression of ifitm induced by h n virus infection or viral particle inoculation. we speculated that the induction of ifitm expression may depend on the interaction between viral particles and cellular factors. moreover, a previous study demonstrated that interactions between cellular factors and envelope glycoprotein b of replication defective human cytomegalovirus may induce isgs expression [ ] . then we verified whether the viral particles were taken up by cells using immunofluorescence. the results showed that viral particles could enter the huvecs (fig. ) , suggesting that cellular interaction might be involved in the induction of ifitm in huvecs. obviously, more work needs to be done to explore this induction mechanism, and data of the present study might considerably narrow the range of possible mechanisms. generally, human influenza virus and avian influenza virus prefer to infect epithelial cells expressing α- , and α- , -linked sialic acid receptors [ , ] . to investigate whether these phenomena were unique responses to h n virus and viral particle in huvecs, we performed the same experiments on epithelial cells synchronously. our results showed that endothelial cells and epithelial cells shared different features. consistent with a previous study [ ] , h n virus infection and viral particle inoculation elevated the levels of type i interferon in beas- bs (fig. ). in addition, contrary to endothelial cells, h n virus infection induced a higher level of ifitm in beas- bs (fig. ) . these data suggested that h n virus or viral particle may stimulate the innate immune response via different ways in vivo. it is of course that further studies are necessary to go beyond our present results. for example, further investigation needs to be done for revealing the precise mechanism of distinct expression patterns of ifitm in epithelial cells and endothelial cells. in particular, whether inactivated viral particle inoculation increases ifitm expression in endothelial cells in vivo and enhances the antiviral state of host. our results showed that inactivated viral particle inoculation increased the expression of ifitm at mrna and protein levels in huvecs. moreover, the induction of ifitm expression mediated the antiviral response in huvecs. characterization of the pathogenicity of members of the newly established h n influenza virus lineages in asia sequence analysis of the hemagglutinin gene of h n korean avian influenza viruses and assessment of the pathogenic potential of isolate ms human infection with an avian h n 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induction of transcriptional repressor protein yy response of airway epithelial cells to double-stranded rna in an allergic environment plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (mdck) in the presence of trypsin porcine reproductive and respiratory syndrome virus counteracts the porcine intrinsic virus restriction factors-ifitm and tetherin in marc- cells transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells characterization and evaluation of monoclonal antibodies developed for typing influenza a and influenza b viruses ifitm inhibits influenza a virus infection by preventing cytosolic entry expression profile and histological distribution of ifitm and ifitm during h n avian influenza virus infection in balb/c mice a morphometric study on the thickness of the pulmonary air-blood barrier global and distinct targets of irf- and irf- during innate response to viral infection irf- /stat [corrected] functional interaction drives retinoic acid-induced gene g expression independently of stat influenza h n virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells essential impact of nf-kappab signaling on the h n influenza a virus-induced transcriptome the broad-spectrum antiviral functions of ifit and ifitm proteins human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein b host immune and apoptotic responses to avian influenza virus h n in human tracheobronchial epithelial cells availability of data and materials all data generated or analyzed during this study are included in this published article.authors' contributions bf participated in the conception and design of the study, carried out most of the experiments and drafted the manuscript. lz and ww analyzed the data. jw and hw carried out part of the experiments. hd and jz modified the manuscript. jq participated in the conception and design of the study, analyzed the data and modified the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - fw xiin authors: si, wei; zhou, shun; wang, zhao; cui, shang-jin title: a multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: fw xiin a multiplex reverse transcription-nested polymerase chain reaction (rt-npcr) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (cdv). a pair of primers (p and p ) specific for cdv corresponding to the highly conserved region of the cdv genome were used as a common primer pair in the first-round pcr of the nested pcr. primers p specific for cdv wild-type strains, were used as the forward primer together with the common reverse primer p in the second round of nested pcr. primers p , p specific for cdv wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer p +p in the second round of nested pcr. a fragment of bp was amplified from vaccine strain genomic rna, and a fragment of bp from wild-type strain genomic rna in the rt-npcr, and two fragments of bp and bp were amplified from the mixed samples of vaccine and wild-type strains. no amplification was achieved for uninfected cells, or cells infected with newcastle disease virus (ndv), canine parvovirus (cpv), canine coronavirus (ccv), rabies virus (rv), or canine adenovirus (cav). the rt-npcr method was used to detect field samples suspected of canine distemper from heilongjiang and jilin provinces, and samples in shandong province. as a result of samples, were found to be wild-type-like, and to be vaccine-strain-like. the rt-npcr method can be used to effectively detect and differentiate wild-type cdv-infected dogs from dogs vaccinated with cdv vaccine, and thus can be used in clinical detection and epidemiological surveillance. canine distemper (cd) is a highly contagious and fatal disease of dogs caused by the canine distemper virus (cdv), which is a single-stranded negative rna virus belonging to the morbillivirus genus within the paramyxoviridae family. other members of the genus include measles virus (mv) and rinderpest virus (rpv). the genome of cdv is approximately , nucleotides (nt) in length, containing several genes encoding n, p, m, f, h, and l proteins. only one serotype has been characterized [ ] . a large number of dogs, minks, foxes die from cdv infections every year, causing significant economic losses [ ] . previous studies [ , ] have reported that vaccinated dogs were infected with cdv in europe and japan. harder et al. also reported that there are marked differences between wild-type and vaccine strains of cdv [ ] , thus whether cdv vaccine strains are able to provide protection from the current strains of cdv remains a question. it is difficult and necessary to discriminate between wild-type and vaccine strains because the attenuated cdv vaccine is used widely in china. so a method to specifically detect the wild-type cdv strains is necessary. the multiplex reverse transcription-nested polymerase chain reaction (rt-npcr) method could be used to effectively detect and differentiate between wild-type cdv-infected dogs from dogs which were vaccinated with cdv vaccine, which would make it useful in clinical diagnosis and epidemiological monitoring. ( '-aaatcctgtgttac-ccgctc- '), p ( '-tggtggctctgcaatatgaa- '), and p ( '-aatgaatggatgcctggggttt- ') were used as the primer specific for cdv species, wildtype strains, and vaccine strain onderstepoort, respectively. primer p ( '-acgtcctggac-cctaagttttg- ') was used as a shared reverse primer. p ( '-ggttttataaaagatt), p ( '-atcta-gaggtaa- ') were used as the primer specific for different cdv vaccine strains. primer pairs p /p , p /p , p /p and p /p were expected to generate a product of , , bp and bp, respectively. cdv vaccine (cdv-a strain) and wild-type strains are maintained in the harbin veterinary research institute. a total of field samples (lung, spleen, liver, or bladder) were collected from different farms in heilongjiang and jilin provinces of china. total rna was extracted from the infected cells with trizol ® reagent in accordance with the manufacturer's instructions. cdna synthesis reaction was performed by polymerase chain reaction as described elsewhere using primer moloney murine leukemia virus reverse transcriptase (m-mlv rt) [ ] [ ] [ ] [ ] [ ] . the first-round pcr was performed with primers p and p . a nested pcr was performed in a total volume of μl containing the firstround pcr products diluted tenfold as well as each of primers p /p , p /p , or p , p /p [ ] [ ] [ ] . the rt-npcr products were visualized by electrophoresis in a % (w/v) agarose gel. rt-npcr was used to detect the cells infected with cdv vaccine strain, wild-type strain, mixed cdv vaccine and wild-type strains, cpv, cav, ccv, rv, ndv, and uninfected cells to test its specificity. extracted rna from serially diluted ( , , , , , - , - , - tcid ) cdv cell cultures ( . tcid /ml) were assayed by rt-npcr to determine its sensitivity. rt-npcr was performed to identify cells infected with cdv vaccine strain, wild-type strain, mixed cdv vaccine and wild-type strains, field samples from heilongjiang and jilin province, cpv, cav, ccv, rv, ndv, and uninfected cells three times to validate the repeatability of the test. samples in heilongjiang and jilin provinces, and fifty-one field samples from dogs, raccoons, foxes, and minks in shandong provinces were assayed by rt-npcr; the background of the field samples is listed in table . all the positive field samples wild-type strain were confirmed by rapid test which bionote, inc. produced. two of the field samples from heilongjiang province were selected for amplification of the h gene of cdv by rt-pcr with primers p ( '-ccaattcatccaagct-gtcc- ') and p ( '-gggatttgaacggttacat-gag- '). the amplified products were cloned and sequenced, and the sequences were aligned with the h genes of a number of cdv strains available in genbank using the megalign function of the dnastar software package. thirty field samples from dogs, foxes, and raccoons in heilongjiang and jilin provinces were also assayed by rt-npcr. after the application of the first-round pcr, primers p and p , and p , p , p and p together, were used to amplify the vaccine and wild-type strains, respectively, at different anneal temperatures. according to the result, when the anneal temperature was from - °c, there was only one specific band. primers p , p , p , p and p were used to perform rt-npcr with different anneal temperatures. only one specific band was observed at an anneal temperature from . - . °c, with the most distinct band appearing at . °c. thus, . °c was chosen for the rt-npcr. a fragment of and bp was amplified from cdv wild-type strain and the vaccine strain, respectively. two bands of and bp were detected simultaneously from the mixed genomic rna of the cdv wild-type and vaccine strains. amplification was not possible for non-cdv viruses, such as cpv, cav, ccv, rv, ndv-infected cells, and uninfected cells by rt-npcr (fig. ) . serial -fold dilutions of cdv vaccine strain were subjected to amplification by multiplex rt-npcr. the lowest limit of detection with this method was shown to be . tcid . of the field samples, tested positive for cdv, among which, showed presence of wild-type viruses, and showed presence of vaccine strain. three independent inter-and intra-assay replicates of the multiplex rt-npcr gave consistent results, indicating the repeatability of the assay. a phylogenetic tree based on the h genes of various cdv strains was generated using the megalign of dnastar software. as shown in fig. , the selected two samples were grouped into wild-type viruses and belonged to a genotype that is obviously different from the cdv vaccine strain. to date, cdv remains one of the most important canine diseases worldwide. surveillance represents a primary concern in the control of cdv. in addition to traditional methods using virus isolation, several promising antigen-elisa, agp, and fa methods have been developed and evaluated. however, these methods are laborious and time consuming. moreover, these tests cannot differentiate cdv wild-type strains from vaccine strains [ , ] . with the recent availability of genomic sequences, molecular diagnostic methods for detection of viruses have significantly improved [ ] . complementary dna and rna probes have been used to detect rna and mrna of the cdv genome with improved specificity and sensitivity [ , ] . primers p and p (specific for cdv and conserved among cdv species), primer p (specific for cdv wild-type strain), and primer p , p (specific for cdv vaccine strain), were selected from the well-conserved regions of the gene encoding matrix protein. a fragment of bp was consistently amplified by rt-pcr with p /p for either cdv vaccine strain or wild-type strain. the nested pcr with p /p generated a bp fragment only for the wild-type strain, while p /p , p / p generated a same bp fragment only for the vaccine strain, and both fragments could be amplified from the mixture of cdv vaccine and wild-type strains. no amplification was obtained for ndv and other common canine viruses, such as ccv, cpv, cav, rv, and uninfected cells control, indicating the high specificity of the method. the method was sensitive, in that it could detect as little as . tcid of the virus. the selected two samples were classified into a branch which belongs to the wild-type strain, but constituting a genotype different from that of cdv vaccine strains, as revealed by phylogenetic analysis based on the sequences of the h gene region, which is believed to be the most reliable classification and genetic typing. rt-npcr was used to detect the field samples in heilongjiang and jilin provinces; of the field samples were cdv-positive, among which were wild-type strain, and were vaccine strain. caideron et al [ ] performed an extensive phylogenetic and molecular evolution analysis on complete sequences of all cdv genes to assess the role of selection and recombination in shaping viral genetic diversity and driving the emergence of cdv in non-dog hosts. they tested the specific hypothesis that molecular adaptation at known receptor-binding sites of the haemagglutinin gene is associated with independent instances of the spread of cdv to novel non-dog hosts in the wild. the selected two samples isolated from dogs were classified into a branch which belongs to the wild-type strain, but constituting a genotype different from that of cdv vaccine strains, as revealed by phylogenetic analysis based on the sequences of the h gene region, which is believed to be the most reliable classification and genetic typing. rt-npcr was used to detect the field samples in shandong provinces; of the field samples were cdv-positive, among which were wild-type strain, were vaccine strain, and were co-infected by wild-type and vaccine strains. in summary, the multiplex rt-npcr developed in this study is a highly specific and sensitive assay for the rapid detection and differentiation of wildtype and vaccine strains of cdv. for lack of other vaccine strains except cdv onderstepoort strain, the primers p and p were designed, but could not use in this time, however, much work needs to be done before a conclusion that this method has an outstanding performance with other cdv vaccine strains. in summary, the multiplex rt-npcr developed in this study is a highly specific and sensitive assay for the rapid detection and differentiation of wild-type and vaccine strains of cdv. animal virology nd edition cd , a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus studies on manifestations of canine distemper virus infection in an urban dog population the nucleotide and predicted amino acid sequence of the fusion protein of recent isolates of canine distemper virus in japan canine distemper virus from diseased large felids: biological properties and polygenetic relationships virus infections of carnivores canine distemper virus infection and encephalitis in javelinas genetic diversity of hungarian canine distemper virus strains comparison of the immunofluorescence assay with rt-pcr and nested pcr in the diagnosis of canine distemper establishment and application of reverse transcription polymerase chain reaction for diagnosis of canine distemper virus. chinese journal of virology detect the pbmc of cdv n gene with rt-pcr. abroad veterinary detection of canine distemper virus (cdv) through one step rt-pcr combined with nested pcr comparison of methods for detecting cdv antibody development of diagnostic method of canine distemper genetic diversity of hungarian canine distemper virus strains detection by rt-pcr and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in argentina a multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus the study was partly supported by funds from the chinese state national hightech r&d program ( program- aa ). we would like to thank dr huaji chou's help to revise the paper. the authors declare that they have no competing interests. authors' contributions ws, sz, zw carried out the experiments and wrote the manuscript. sc conceived the studies and participated in experimental design and coordination. all authors read and approved the final manuscript. key: cord- -zf jbhdl authors: he, zhongping; zhuang, hui; zhao, chunhui; dong, qingming; peng, guoai; dwyer, dominic e title: using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (sars-cov) date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: zf jbhdl background: severe acute respiratory syndrome (sars) caused a large outbreak of pneumonia in beijing, china, in . reverse transcriptase polymerase chain reaction (rt-pcr) was used to detect and quantify sars-cov in sera and self-collected throat washes and fecal samples from patients with laboratory-confirmed sars managed at a single institution. results: sars-cov detection rates in sera were highest in the first days of illness, whereas detection was highest in throat washes – days after onset of symptoms. the highest sars-cov rt-pcr rates ( . – . %) and viral loads (log( ) . – . ) were seen in fecal samples collected – weeks after the onset of clinical illness. fecal samples were frequently sars-cov rt-pcr positive beyond days, and occasional sera still had sars-cov detected after weeks of illness. conclusion: in the context of an extensive outbreak with major pressure on hospital resources, patient self-collected samples are an alternative to nasopharyngeal aspirates for laboratory confirmation of sars-cov infection. severe acute respiratory syndrome (sars) emerged in late , with more than cases reported by april by the world health organization, mostly in china ( ), taiwan, hong kong sar, singapore and canada. there were deaths and a mortality of . % [ ] . the largest outbreak was in beijing, with over , cases [ ] . the sars-associated coronavirus (sars-cov) was identified as the causal agent following its isolation and detec-tion by electron microscopy and reverse transcriptase polymerase chain reaction (rt-pcr) from a range of clinical specimens. serological evidence of infection has been found in most patients fitting the clinical definition of sars [ ] [ ] [ ] [ ] . the clinical, radiological, and laboratory findings of sars from beijing and elsewhere have been described previously [ , , [ ] [ ] [ ] [ ] [ ] [ ] . the aim of this study was to detect and quantify sars-cov using rt-pcr in sera and throat washes and stools self-collected by patients with laboratory confirmed sars managed at a single institution. these samples were collected during the extreme pressure of the beijing sars outbreak in the context of healthcare worker concern about the safety of collecting nsaopharyngeal aspirates (npas) from ill patients. between march -may , patients fitting the case definition of probable sars were hospitalized. of these, were laboratory confirmed following the detection of sars-cov-specific igm and/or igg antibody by immunofluorescence [ ] and/or by the detection of sars-cov rna by rt-pcr. the mean age of the cohort was ± years. there were ( . %) healthcare workers who acquired sars, including nurses, physicians, logistics staff, pharmacists and laboratory technicians (one of whom was believed to be infected after handling sputum and stool samples from sars patients in a diagnostic laboratory). a total of people were infected following exposure to sars patients in the hospital setting, either as healthcare workers, patients or visitors, and another cases were household contacts of known sars cases. common clinical features on admission included fever ( %), subjective shortness of breath ( %), nonproductive cough ( %), malaise ( %), myalgia ( %), headache ( %), dyspnea ( %), chills ( %), diarrhea ( %) and sore throat ( %). the mortality rate was . % ( / ) amongst laboratory-confirmed cases. sera, throat washes and stool samples were tested for sars-cov rna by rt-pcr. a total of sera (ranging from - per patient) were collected - days after the onset of illness from cases. overall, . % ( / ) of sera had detectable amounts of sars-cov rna detected, with viral loads ranging from - copies/ml serum (table ) . sera collected within days of disease onset were more likely to be rt-pcr positive ( %) than later in the disease course, although sars-cov rna was still occasionally detected in sera out to days of illness. a single throat wash was self-collected by patients to days after the onset of disease. a total of ( . %) had sars-cov rna detected by rt-pcr (table ) , with viral loads ranging from - copies/ml wash fluid. the highest detection rate was % in throat washes collected between days and . of stool samples self-collected by patients ( - samples each), ( . %) had sars-cov rna detected by rt-pcr (table ) . stool samples were not collected in the first days of illness, but high rates of sars-cov rna detection ( / , . %) were seen in stools collected between and days after onset. viral loads in stool were as high as copies/g feces from day . fecal samples collected days or more after onset of disease contained sars-cov rna in . % ( / ) of samples, with a mean load of copies/g feces. the fecal load of sars-cov was at least between and logs higher than in throat washes or sera at comparable time points. the clinical features of this cohort of patients managed at a single institution were similar to those reported elsewhere [ , , [ ] [ ] [ ] [ ] [ ] [ ] , although diarrhea was present in only % of patients compared to rates of - % reported in studies from hong kong and canada [ ] [ ] [ ] . like other sars outbreaks, many cases ( . %) were acquired after exposure in the hospital environment, with healthcare workers providing % of cases at this institution. of note was a case of sars possibly acquired in a diagnostic laboratory. there have since been a number of cases acquired in research laboratories [ ] . detection of sars-cov rna by rt-pcr is only moderately useful in the early diagnosis of sars, as the maximal viral load and rt-pcr sensitivity occurs in the second week of illness [ ] . in addition, the sensitivity of sars-cov rt-pcr on specimens collected from different sites and at different time points in the illness varies. testing more than one clinical specimen increases the likelihood of obtaining a positive rt-pcr result. in one large study, % of patients with clinical sars had a positive sars-cov rt-pcr in one or more clinical specimens, with the highest detection rates in sputum ( . %), npas ( . %) and nose/throat swabs ( %) collected within the first days of illness [ ] . we found that the likelihood of a positive sars-cov rt-pcr was similar in serum ( . %) and throat washes ( . %) in the first days of illness. we found the peak of sars-cov detection in throat washes to be between days to , where . % ( / ) of samples were positive, similar to reported rates in other respiratory specimens [ , ] . the viral loads in throat washes decreased over time and were at levels between those in feces and sera at similar time points. in other studies, throat swabs were rt-pcr positive in . % of probable sars cases, reaching - % on days - , and consistent with earlier studies showing peaks of virus shedding in the respiratory tract in the second week of illness [ , ] . high viral loads were seen in npas in patients with sars, mainly in the second week of illness [ ] . the use of patient self-collected throat washings may reduce risks to healthcare workers, although lower respiratory tract samples such as sputum, npas or bronchoalveolar lavage fluid are likely to have higher viral loads and offer increased likelihood of sars-cov detection by rt-pcr. we were unable to correlate viral loads in the various clinical samples with ability to isolate virus or transmission to other people; whether viral load in the respiratory tract correlates with 'super-shedding' events is uncertain. although overall sars-cov detection rates and viral load in throat washes and stools were higher than in the serum, serum sars-cov rt-pcr is a useful investigation early in the illness as we found that % of sera had sars-cov detected in the first four days of illness. one study of sera from probable sars patients found a detectable sars-cov load ranging from × to × copies/ml serum in % of the samples, but not after days after onset [ ] . of interest was that occasional serum samples from individuals remained sars-cov rt-pcr positive (with moderate viral loads) over three weeks after onset of illness, a feature noted in another study [ ] . high rates of sars-cov rt-pcr detection (as high as . % between days - ) and high viral loads were found in fecal samples in the second to fourth weeks of disease. rates of sars-cov detection in fecal samples began to decrease after one month, although many stools were still sars-cov rna positive days or more after the onset of the clinical illness. the sars-cov load in fecal samples collected after days were higher than the peak load seen in sera collected early in disease, and comparable to the viral load in throat washes in the second week of illness. both lower ( % in fecal samples collected - days after onset) and similar high detection rates (over % in stools collected - days after onset) have been reported elsewhere, as have fecal samples posi-tive days or more after onset [ , ] . despite the high sars-cov load in feces, diarrhea was not a prominent clinical feature in this cohort. long-term fecal viral shedding may be an additional source of community spread of sars, although the infectivity of feces may be better assessed with virus isolation. direct comparisons of the sensitivity and specificity of rt-pcr for the detection of sars-cov are hampered by the use of different types of clinical specimens, rna extraction procedures and different rt-pcr techniques. the first published interlaboratory comparison showed sensitivities of % and % for npas, % and % for throat swabs, % and % for urine samples and % and % for stool specimens, with an overall specificity of % [ ] . to date, no significant differences in the sensitivity and specificity of various commercial and in-house rt-pcr or other molecular assays have been reported [ ] [ ] [ ] . it causes significant nosocomial infection and requires aggressive infection control practices rarely used for other causes of atypical pneumonia. laboratory confirmation of sars is crucial in the management of patients presenting with pneumonia, particularly as the clinical features of sars make it difficult to distinguish from other causes of atypical pneumonia. molecular methods for sars diagnosis are useful, although their value is affected by the observation that maximal viral shedding occurs after the first week of illness rather than at the initial clinical presentation. the sars outbreak was characterized by high infection rates in healthcare workers; patient self-collected specimens such as throat washes or feces, or serum may pose less risk to healthcare workers, particularly in the context of concerns about nosocomial acquisition. although npas and other lower respiratory tract samples are the sample of choice for suspected respiratory viral infections, patient self-collected specimens are suitable for rt-pcr. thus they offer diagnostic value, especially in sars where the peak of viral shedding is after the first week of illness, and this sampling approach may reduce the safety issues of healthcare workers collecting npas. patient self-collected specimens may be less appropriate for common seasonal respiratory virus infections such as influenza, where viral shedding is maximal at clinical presentation and virus is rarely detected outside the respiratory tract. accurate and rapid laboratory diagnosis will become even more important as sars becomes less common, or in the event of new outbreaks of sars, especially if influenza or other seasonal respiratory viruses are co-circulating. this study was conducted during the first three months (march-may ) of the sars outbreak in beijing, china, where ditan hospital was designated as a 'sars hospital', meaning that suspected sars patients were transferred and managed at this institution. the study was approved by the ethics committee of the beijing ditan hospital. the clinical case definition of probable sars included a fever of ≥ °c, cough or shortness of breath, new pulmonary infiltrates on chest radiography, and close contact with a suspect or probable sars case. day was defined as the day of fever onset. sera, throat washes and feces were collected from hospitalised patients for testing with a quantitative sars-cov rt-pcr. as the early phase of the outbreak in beijing had involved many healthcare workers [ ] , patient self-collected throat washes and fecal samples were used to minimize further nosocomial transmission. for throat washes, patients were given ml of sterile . % nacl, asked to gargle for seconds then spit the fluid into a ml sterile plastic screw-topped plastic container. patients were also asked to collect approximately cm feces and place it into a ml sterile screw-topped plastic container. five ml of the throat wash was centrifuged at , g for minutes, then the supernatant further centrifuged at , g for hour. ten ml of . m phosphate buffered saline (ph . ) was used to dilute the fecal sample, then ml was centrifuged at , g for minutes. the supernatant was further centrifuged at , g for hour. rna was extracted from the remaining - μl of the throat wash and fecal pellets using trizol (invitrogen, beijing, pr china). μl of sera were centrifuged at , g for hour, the supernatant removed and rna extracted from the remaining - μl pellet using trizol. sars-cov rna was detected in throat washes, stool and blood using a fluorescence quantitative rt-pcr assay (shenzhen pj biotech company, shenzhen, guangdong province, pr china), according to the manufacturer's instructions and performed on a biorad icycler thermal cycler (bio-rad laboratories, beijing, pr china). the sars-cov pol region primers used were p sense 'gttcttgctcgcaaacataacactt ' (position - in sars-cov urbani strain, genbank accession number ay ), p antisense 'aacagcttgacaaatgttaaagaca ' ( - ) and probe 'tgtgtggcggctcactatat ' ( - ). internal controls were used in all runs, and no evidence of pcr inhibition in clinical samples was detected. testing for other respiratory viruses was not carried out in this cohort of patients as they fitted the sars clinical case definition during the outbreak. the pcr assay was negative when performed on rna or dna extracted from influenza a and b, rhinovirus, respiratory syncytial virus and adenovirus isolates, and on plasma collected from otherwise healthy hepatitis c and b infected individuals (data not shown). manipulations were carried out in a bsl facility with bsl practices. sars-cov isolation was not attempted on clinical samples during the outbreak due to safety concerns and time constraints. the author(s) declare that they have no competing interests. the study was funded by the beijing ditan hospital. organization wh: cumulative number of reported probable cases of severe acute respiratory syndrome (sars) for the beijing joint sars expert group: severe acute respiratory syndrome group sarsw: a novel coronavirus associated with severe acute respiratory syndrome identification of a novel virus in patients with severe acute respiratory syndrome and members of the sars study group: coronavirus as a possible cause of severe acute respiratory syndrome kinetics of severe acute respiratory syndrome (sars) coronavirus-specific antibodies in laboratory confirmed cases of sars clinical features and short-term outcomes of patients with sars in the greater toronto area a major outbreak of severe acute respiratory syndrome in hong kong and members of the hku/uch sars study group: clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars coronavirus in patients with suspected sars. emerging infectious diseases serologic and molecular biologic methods for sars-associated coronavirus infection a clinicopathological study of three cases of severe acute respiratory syndrome (sars) detection of severe acute respiratory syndrome coronavirus in blood of infected patients real-time polymerase chain reaction for detecting sars coronavirus evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus ontario laboratory working group for the rapid diagnosis of emerging infections: performance and cost evaluation of one commercial and six in-house conventional and real-time reverse transcription-pcr assays for detection of severe acute respiratory syndrome coronavirus rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification clinical evaluation of real-time pcr assays for rapid diagnosis of sars coronavirus during outbreak and post-epidemic periods zh and dd reviewed the data and wrote the manuscript. zh, qd, cz, gp, and hz collected the samples and clinical data, and undetook the performance of the molecular assays. dd was a short-term consultant with the world health organisation in beijing, china during the sars outbreak in . key: cord- -paucodwz authors: finkbeiner, stacy r; kirkwood, carl d; wang, david title: complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: paucodwz background: astroviruses infect a variety of mammals and birds and are causative agents of diarrhea in humans and other animal hosts. we have previously described the identification of several sequence fragments with limited sequence identity to known astroviruses in a stool specimen obtained from a child with acute diarrhea, suggesting that a novel virus was present. results: in this study, the complete genome of this novel virus isolate was sequenced and analyzed. the overall genome organization of this virus paralleled that of known astroviruses, with open reading frames identified. phylogenetic analysis of the orfs indicated that this virus is highly divergent from all previously described animal and human astroviruses. molecular features that are highly conserved in human serotypes – , such as a 'ntr stem-loop structure and conserved nucleotide motifs present in the 'ntr and orf b/ junction, were either absent or only partially conserved in this novel virus. conclusion: based on the analyses described herein, we propose that this newly discovered virus represents a novel species in the family astroviridae. it has tentatively been named astrovirus mlb . astroviruses are non-enveloped, single stranded, positive sense rna viruses. their genomes range from approximately to kb in length, are polyadenylated, and have both ' and ' non-translated regions (ntr) [ ] . their genomes have three open reading frames (orfs) organized from ' to ' as follows: orf a, which encodes a serine protease; orf b, which encodes the rna dependent polymerase; and orf , which encodes the structural proteins. a frameshift must occur during the translation of orf a in order for orf b to be translated. orf is translated from a sub-genomic rna and produces a polyprotein which is cleaved by cellular proteases [ ] . the family astroviridae includes closely related human serotypes as well as additional members that infect cattle, sheep, cats, dogs, deer, chickens, turkeys, and ducks [ ] . although some of the animal astroviruses are known to cause hepatitis or nephritis [ ] , astroviruses typically cause diarrhea in their hosts. human astrovirus infections most frequently cause watery diarrhea lasting - days, and less commonly vomiting, headache, fever, abdominal pains, and anorexia in children under the age of , the elderly, and immunocompromised individuals [ ] . the known human astroviruses account for up to ~ % of sporadic cases of non-bacterial diarrhea in children [ ] [ ] [ ] [ ] [ ] . diarrhea is the third leading infectious cause of death worldwide and is responsible for approximately million deaths each year as well as [ ] an estimated . billion non-fatal episodes [ , ] . in children, rotaviruses, caliciviruses, adenoviruses and astroviruses are responsible for the greatest proportion of cases [ , , [ ] [ ] [ ] . most epidemiological studies fail to identify an etiologic agent iñ % of diarrhea cases [ ] [ ] [ ] [ ] [ ] . recently, we conducted viral metagenomic analysis of diarrhea samples using a mass sequencing approach with the explicit goal of identifying novel viruses that may be candidate causes of diarrhea. one of the stool samples we analyzed was collected in at the royal children's hospital in melbourne, australia from a -yr old boy with acute diarrhea. seven sequence reads were identified in this sample that shared ≤ % amino acid identity to known astrovirus proteins, suggesting that a novel astrovirus was present in the sample [ ] . in this paper, we report the full sequencing and characterization of the genome of this astrovirus, referred to hereafter as astrovirus mlb (astv-mlb ). in the previous metagenomic study [ ] , we identified seven sequence reads with limited identity to known astroviruses that could be assembled into two small contigs in a clinical stool sample. the contigs had - %, and - % amino acid identity to human astrovirus serine proteases and rna-polymerases, respectively. in this study, the complete genome of the astrovirus present in the original stool specimen was sequenced to an average of > × coverage [genbank: fj ]. the virus has been tentatively named astrovirus mlb (astv-mlb ). analysis of the genome showed that astv-mlb has the same genomic organization as other astroviruses. like other astroviruses, the astv-mlb genome was predicted to encode three open reading frames (orf a, orf b, and orf ) and contained both ' and ' non-translated regions (ntr), as well as a poly-a tail. the complete genome length of astv-mlb was , bp, excluding the poly-a tail, slightly shorter when compared to other astrovirus genomes which range in size between ~ , and , bp [ ] . a comparison of astv-mlb genomic elements with those of fully sequenced astroviruses is shown in table . the orf a of astroviruses encodes a non-structural polyprotein which contains a serine-like protease motif. pfam analysis revealed a region of orf a that has homology to a peptidase domain. in addition, alignment of astv-mlb with other astroviruses revealed that astv-mlb contains the amino acids of the catalytic triad (his, asp, ser) which are conserved in the c-like protease motif found in other viruses (data not shown) [ ] . the residues rtq which have been suggested to be involved in substrate binding are conserved among the human astroviruses, but vary in other viruses which have the c-like motif [ ] . in astv-mlb , the predicted substrate binding residues (atr) are identical to those found in ovine astrovirus and not those of the human astroviruses (data not shown). a second feature of astrovirus orf a is the presence of a bipartite nuclear localization signal (nls) found in human, chicken, and ovine astroviruses, but not turkey astroviruses [ ] . a bipartite nls is characterized as having two regions of basic amino acids separated by a aa spacer. the protein alignment of orf a revealed that astv-mlb has a sequence motif similar to the putative nls of human astroviruses. this region of the genome has also been predicted to potentially encode for a viral genome-linked protein (vpg) [ ] . the high sequence similarity observed between astv-mlb and other astroviruses in the motifs identified as essential for a putative vpg suggests that astv-mlb may also encode a vpg (data not shown). while no experimental data exists supporting the prediction of the presence of a vpg being encoded in any of the astrovirus genomes, we should note that we did encounter difficulty in obtaining the ' end of the mlb genome until treatment of the rna with proteinase k prior to rna extraction was added to the experimental protocol. finally, the , nt sequence of astv-mlb orf a is shorter than orf a sequences of other astroviruses, which range between ~ , - , nt ( table ). the shorter length of astv-mlb orf a relative to the human astroviruses is largely attributable to two deletions totaling amino acids located within a highly conserved motif near the carboxyl terminus of human astroviruses - . this deletion falls within a aa region that has been mapped as being an immunoreactive epitope in human astroviruses [ ] and is located in the non-structural protein p [ ] . recently, p has been reported to lead to apoptosis of the host cell which results in efficient virus replication [ ] and particle release [ ] . however, it is unclear how the genome deletion identified in astv-mlb might influence these activities. astrovirus orf b is classically generated by a - ribosomal frameshift induced by the presence of a heptameric 'slippery sequence' (aaaaaaac). [ ] . a conserved slippery sequence was identified near the end of orf a of ast-mlb and fsfinder was used to determine if the downstream sequence was capable of forming a stemloop structure, as found in other astoviruses [ ] . the predicted start position of orf b was then determined by selecting the first amino acid in frame with the slippery sequence. the b open reading frame of astroviruses encodes an rna-dependent rna polymerase (rnap). pfam analysis revealed that astv-mlb orf b contains the rna-dependent rna polymerase domain found in other positive strand rna viruses, suggesting this orf does in fact encode for an rnap. astrovirus orf encodes a large structural polyprotein that is cleaved by cellular proteases to generate the viral capsid proteins. following the convention of human astroviruses [ , ] by choosing a start codon for orf located two nucleotides upstream of the orf b stop codon resulted in a predicted protein length of aa. pfam analysis of the predicted protein encoded by orf identifies an astrovirus capsid motif, thereby congruent with the paradigm of astrovirus genome organization in which orf encodes the structural capsid proteins. the astv-mlb orf protein sequence was divided into four subregions for more detailed analysis as described [ ] . pair-wise comparisons of each region were conducted between the astv-mlb sequence and the sequences of all astroviruses for which sequences were available. consistent with previous reports, region i appeared to be the most conserved of the four regions and in each of the regions, astv-mlb shared the most similarity to known human astroviruses. however, even in region i, astv-mlb only exhibited - % identity to known human astroviruses. in the less conserved regions ii-iv, astv-mlb shared only - % amino acid identity to the known human astroviruses. by contrast, the range of identities between human astrovirus serotypes - were, - %, - % and - % for regions ii, iii and iv, respectively. overall, astv-mlb maintained higher conservation in region i of orf than in other regions, consistent with paradigms established by analysis of other astroviruses. multiple independent ' race experiments were performed to determine the precise ' end of the genome. based on these experiments, the astv-mlb ' ntr was determined to be nt long. this is similar in length to the ~ - nt 'ntrs of avian astroviruses [ ] , but much shorter than the - nt long 'ntrs of the human astrovirus serotypes (table ) . notably, the human astroviruses share a nt consensus sequence at the terminal ' nucleotides of the genome which is not conserved in other astroviruses (data not shown). astv-mlb contained out of the consensus nucleotides, including the most 'ccaa motif within the this region [ ] (fig. a) . these data support the notion that the sequence we generated does contain the very ' terminus of the genome. multiple sequence alignments of putative astrovirus regula-tory regions human astroviruses contain a nt region at the junction between orf b and orf that is ~ - % conserved between serotypes [ ] . the most highly conserved core nt region of this sequence is - % identical among the human astrovirus serotypes. the exact role of this sequence is not known, but it is hypothesized to be a regulatory element of the sub-genomic rna that encodes for orf . alignment between astv-mlb and other human astroviruses of the highly conserved nt at the orf b/orf junction revealed that astv-mlb possessed only . % identity in this region (fig. b) . by contrast, the known animal astroviruses share only - . % identity in this nt region with human astroviruses as determined by pair-wise comparisons. interestingly, astv-mlb shares . % identity in this region to ovine astrovirus. all of the previously described astroviruses, with the exception of turkey astrovirus , have a conserved rna secondary structure referred to as the stem-loop ii-like motif (s m) found at the ' end of the genome in the ' ntr [ ] . this motif is also present in some coronaviruses and equine rhinovirus serotype . mutations within this motif are generally accompanied by compensatory mutations that restore base pairing [ ] . the conservation of such a sequence motif across multiple viral families suggests that it may play a broad role in the biology of positive stranded rna viruses [ ] . the exact function of this stem loop is not known, but it is hypothesized to interact with viral and cellular proteins needed for rna replication. nucleotide alignment of the nucleotides at the ' terminus of the astv-mlb genome and other viruses known to contain the stem-loop motif suggested that astv-mlb does not have this conserved nucleotide motif (data not shown). furthermore, it also has the shortest 'ntr reported to date for an astrovirus. (table ) [ ] . multiple sequence alignments of the three astrovirus open reading frames were performed and bootstrapped maximum parsimony trees were generated (fig. ) . the trees confirmed initial assessments that astv-mlb is a novel astrovirus [ ] . the trees for orfs a and b (fig. a, b) both indicated that astv-mlb is most closely related to the human astroviruses, although it is highly divergent from them. astv-mlb orf a only has - % amino acid identity to other astrovirus orf a proteins and the pairwise sequence alignments of orf b revealed - % amino acid identity between orf b proteins of astv-mlb and other astroviruses ( table ). the maximum parsimony tree for orf (fig. c) shows that there is greater divergence among all of the sequences for orf , as is to be expected of the capsid region. however it is still evident that astv-mlb is quite divergent from any of the known human astroviruses. based on the predicted aa protein of orf , astv-mlb has only - % amino acid identity to other astrovirus capsid precursor proteins ( table ). at this point, the origin of astv-mlb is unclear. astv-mlb may be a bona fide human virus capable of infecting and replicating within the human gastrointestinal tract that had evaded detection until now. alternately, it may be a passenger virus present simply as a result of dietary ingestion, as has been described previously for plant viruses detected in human stool [ ] . of course, viruses derived from dietary intake that appear to cause human disease, such as aichi virus, have been described previously [ , ] . another possibility is that this virus may represent zoonotic transmission from some other animal species that is the true host for astrovirus mlb . traditionally it has been thought that astroviruses have a strict species tropism. however, recent evidence has emerged that suggests that interspecies transmission does occur. for example, chicken astrovirus antibodies have been detected in turkeys [ ] and an astrovirus was isolated from humans whose capsid sequence most closely resembled that of feline astrovirus [ ] . because of the uncertainty as to the identity of the true host species and the host range for this virus, we have tentatively named this novel virus astrovirus mlb (astv-mlb ). efforts to define whether astv-mlb is a novel human pathogen are underway. complete sequencing and genome analysis of astrovirus mlb revealed that the virus has three open reading a na na na na b na na na na frames sharing the same organization as other astroviruses. phylogenetic analysis of the open reading frames clearly demonstrated that astv-mlb is highly divergent from any of the known astroviruses. furthermore, astv-mlb lacks the conservation seen between human astroviruses - in the non-translated regions of the genome such as the ' and ' ntr and the orf b/ junction. the aggregate analysis of the non-coding features and orfs as well as the phylogentic analysis clearly indicates that astv-mlb is highly divergent from all previously described astroviruses. the divergence of astv-mlb from known astroviruses in the non-translated regions of the genome is particularly interesting because these regions are nucleotide motifs that are thought to play regulatory roles in viral replication. this suggests that astv-mlb may behave very differently from the known astroviruses and that additional studies on the regulation of astv-mlb transcription and replication may broaden our understanding of astrovirus paradigms. astroviruses are associated with diarrhea predominantly in young children and immunocompromised individuals. the discovery of astv-mlb in a liver transplant patient fits well with the known clinical parameters of astrovirus infection. we previously reported that the only other virus detected in this stool was a tt virus [ ] , which is thought to be non-pathogenic [ ] . it is therefore tempting to speculate that astv-mlb is the pathogenic agent that caused this case of diarrhea. however, whether astv-mlb is a bona fide human virus capable of causing diarrhea will have to be established by further experimentation and epidemiological surveys. a stool sample was collected from a year old boy admitted to the royal children's hospital with acute diarrhea in . the child had previously undergone a liver transplant one year prior to this episode of diarrhea, however the immunological status was unknown. rna was isolated from the primary stool filtrate using rna-bee (tel-test, inc.) according to manufacturer's instructions. in some cases, the stool filtrate was treated with . mg\ml proteinase k (sigma) for min prior to rna extraction. the astrovirus sequence reads previously detected in the primary stool filtrate [ ] [genbank accessions: et , et , et , et , et , et , et ] were assembled into two contigs, and the nucleic acid between the contigs was obtained by rt-pcr. for reverse transcription reactions, cdna was generated with monsterscript rt at °c and amplified with taq (invitrogen). subsequent ' and ' race reactions were done to obtain the entire genome. to generate high quality sequence coverage, pairs of specific primers that spanned the complete genome in overlapping ~ kb fragments were used in rt-pcr reactions and then cloned and sequenced using standard sanger sequencing chemistry. all amplicons were cloned into pcr . (invitrogen). these primer pairs were used to confirm the sequence of the viral genome from both the primary stool sample and the passage tissue culture sample. the complete genome sequence of astv-mlb has been deposited in [genbank: fj ]. open reading frames a and were predicted for astv-mlb using the ncbi orf finder program. orf b was predicted based on the frameshift paradigm that occurs in other astroviruses by identifying a heptameric slippery sequence [ ] . conserved motifs were identified using pfam [ ] . bioedit was used to determine the percent identity between sequences as determined by pair-wise alignments. clustalx ( . ) was used to carry out multiple sequence alignments of the protein sequences associated with all three of the open reading frames of representative astrovirus types. maximum parsimony trees were generated using paup with , bootstrap replicates [ ] . available nucleotide or protein sequences of the following astroviruses were obtained pathogenesis of astrovirus infection the changing epidemiology of astrovirus-associated gastroenteritis: a review diarrhea etiology in a children's hospital emergency department: a prospective cohort study a -year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in astrovirus detection in sporadic cases of diarrhea among hospitalized and non-hospitalized children in rio de janeiro, brazil, from to detection of sporadic cases of norovirus infection in hospitalized children in italy a millennium update on pediatric diarrheal illness in the developing world the global burden of diarrhoeal disease diarrheagenic escherichia coli infection in a review of viral gastroenteritis viruses causing gastroenteritis importance of a new virus in acute sporadic enteritis in children astrovirus infection in volunteers pattern of shedding of the norwalk particle in stools during experimentally induced gastroenteritis in volunteers as determined by immune electron microscopy isolation and identification of enteric adenoviruses metagenomic analysis of human diarrhea: viral detection and discovery proteolytic processing of a human astrovirus nonstructural protein complete genomic sequences of astroviruses from sheep and turkey: comparison with related viruses genome prediction of putative genome-linked viral protein (vpg) of astroviruses cloning and characterization of human astrovirus immunoreactive epitopes apoptosis in astrovirus-infected caco- cells caspases mediate processing of the capsid precursor and cell release of human astroviruses predicting genes expressed via - and + frameshifts subgenomic rna sequence of human astrovirus supports classification of astroviridae as a new family of rna viruses identification and sequence determination of the capsid protein gene of human astrovirus serotype ushijima h: genetic analysis of the capsid region of astroviruses molecular analysis of a serotype human astrovirus genome molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children molecular characterisation of the '-end of the astrovirus genome rna viral community in human feces: prevalence of plant pathogenic viruses prevalence of newly isolated, cytopathic small round virus (aichi strain) in japan isomura s: isolation of cytopathic small round viruses with bs-c- cells from patients with gastroenteritis the isolation and characterisation of astroviruses from chickens molecular properties, biology, and clinical implications of tt virus, a recently identified widespread infectious agent of humans rna sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis pfam: clans, web tools and services phylogenetic analysis using parsimony (*and other methods). version th edition this work was funded in part by an nhmrc rd wright research fellowship (id , ck), and by the food safety research response network, a coordinated agricultural project, funded through the national research initiative of the usda cooperative state research, education and extension service, grant number ## - - . the authors declare that they have no competing interests. dw conceived and designed the experiments. sf carried out the experiments and analysis. ck contributed reagents/materials. sf and dw wrote the paper. key: cord- -yyz z xj authors: abdel-moneim, ahmed s; zlotowski, priscila; veits, jutta; keil, günther m; teifke, jens p title: immunohistochemistry for detection of avian infectious bronchitis virus strain m in the proventriculus and nervous system of experimentally infected chicken embryos date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: yyz z xj background: infectious bronchitis virus primarily induces a disease of the respiratory system, different ibv strains may show variable tissue tropisms and also affect the oviduct and the kidneys. proventriculitis was also associated with some new ibv strains. aim of this study was to investigate by immunohistochemistry (ihc) the tissue tropism of avian infectious bronchitis virus (ibv) strain m in experimentally infected chicken embryos. results: to this end chicken embryos were inoculated in the allantoic sac with ( )eid( )of ibv m at days of age. at , , and h postinoculation (pi), embryos and chorioallantoic membranes (cam) were sampled, fixed, and paraffin-wax embedded. allantoic fluid was also collected and titrated in chicken embryo kidney cells (cek). the sensitivity of ihc in detecting ibv antigens in the cam of inoculated eggs matched the virus reisolation and detection in cek. using ihc, antigens of ibv were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. these results were consistent with virus isolation and denote the wide tissue tropism of ibv m in the chicken embryo. most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with ibv strain m . conclusion: ihc can be an additional useful tool for diagnosis of ibv infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with ibv strains of different virulence. moreover, these results underline that embryonic tissues in addition to cam could be also used as possible source to generate ibv antigens for diagnostic purposes. infectious bronchitis virus is the prototype species of the family coronaviridae in the order nidovirales. more than genotypes are distributed worldwide. ibv causes an acute highly contagious viral respiratory disease of chickens which is characterized by respiratory rales, coughing and sneezing [ ] . some ibv strains replicate in the gastrointestinal tract, oviduct, and kidney, and due to their nephropathogenic properties they have the potential to cause severe losses with up to % mortality [ , ] . in other cases, infection of the proventriculus leads to % to % mortality in young birds [ ] . most isolates of ibv replicate well in the developing chicken embryo following inoculation of the allantoic cavity, and high titers of virus can be isolated from the allantoic fluid [ ] . replication of ibv strains m and beaudette in vitro is restricted to primary chicken cells and depends on the expression of , linked sialic acids on the cell surface. thus, these molecules are supposed to serve as receptor determinants for primary attachment of ibv to host cells [ , ] . the conventional diagnosis of the ibv is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. since two or three blind passages are often required for successful primary isolation of ibv, this procedure could be tedious and time consuming. alternatively, ibv may be isolated by inoculation in chicken tracheal organ cultures. this method is sensitive [ ] but highly laborious. furthermore, ibv may be detected directly in tissues of infected birds by means of immunohistochemistry [ , ] or in situ hybridization [ ] . the reverse transcription-polymerase chain reaction (rt-pcr) has proved useful in the detection of several rna viruses [ ] . aim of this study was first to evaluate the suitability of ihc for detection of ibv antigen in paraffinwax embedded cam and second to analyse the viral antigen distribution in different embryonic tissues between and h after experimental infection. inoculation of eid ibv m in spf ece resulted in death of embryos at h ( embryos), h ( embryos) and single embryonic death at , and h after ibv inoculation. allantoic fluid and cam were harvested at the times given in table which also showed that high virus titers were obtained - h pi that decreased sharply to tcid at h pi (table ) . immunostaining of the cam was positive in all inoculated eggs from which infectious virus was also recovered. only one embryo showed non specific death at h pi as it showed negative results in both immunostaining and virus recovery assays. ihc detected infected cam that possesses virus titers of only or tcid in the corresponding allantoic fluid (table ) . ibv antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos (table , figure ). moderate number of positive cells (++) were detected in the mucosa, smooth muscle fibres and vasculature of gizzard at , , and h pi. few (+) to moderate (++) number of positive cells were detected in the proventriculus mucosa as well as its smooth muscle fibres in embryos dead at , and h pi ( figure a ). positive immunostaining was also detected in macrophages of both spleen ( , , , , h pi) ( figure c ) and liver (kupffer cells) ( , , , h pi). ibv antigens were also present in neurons of both spinal cord ( and h pi) and brain ( and h pi) ( figure b) , heart ( , , h pi) ( figure d) classic methods for ibv diagnosis include serological tests for analysis of antibody titers against ibv [ ] and virus isolation in embryonated chicken eggs since ibv grows well in the developing chicken embryo. these methods are inherently slow and time consuming. currently, detection and serotype analysis of ibv is performed by rt-pcr and restriction fragment length polymorphism analysis [ ] or by sequencing rt-pcr products of s gene [ ] . to establish ihc on paraffin-wax embedded tissues using a polyclonal rabbit anti-ibv serum, embryonated eggs were inoculated with eid ibv m . cam and embryos were collected till h pi, since the presence of ibv antigen in inoculated eggs by an antigen detection method is preferably performed to days after inoculation [ ] . this also confirmed in the current study where ibv titers declined sharply at h pi. it is well known that maximum ibv virus titers reached to days postinoculation [ ] [ ] [ ] but interestingly, allantoic fluid showed high virus titers - h pi. immunostaining of the cam was positive in all inoculated eggs from which infectious virus was recovered. it is worth to note that also samples of infected cam with respective allantoic fluid virus titers of only or tcid were obtained, stained positively with the ibv antiserum which equals approximately or eid [ ] . this indicates a relatively high sensitivity of the immunohistochemistry applied in this study. using a monoclonal antibody for immunostaining, the detection limit for ibv antigen was . eid as described by [ ] . these differences in sensitivity may be due to larger number of antigenic epitopes recognized by the polyclonal serum. this finding prompted us to apply ihc for screening of antigen distribution in different tissues in the ibv inoculated embryos. antigens were detected in the nasal epithelium, trachea, lung, spleen, myocardium, liver, gizzard, proventriculus, kidney, skin, sclera of the eye, spinal cord, as well as in neurons of the central nervous system in infected embryos. however, isolation or detection of virus by virological methods may indicate only that virus is present in the tissue due to viraemia and thus does not necessarily prove productive replication in the respective organs. hence, tissue tropism can not be determined by virus isolation or detection only [ ] but requires morphologically based techniques. although chong & apostolov [ ] , failed to detect virus by immunofluorescence in the intestine and cecal tonsils of chickens experimentally infected with m of ibv, lucio & fabricant [ ] found that m can infect a variety of tissues and that some isolates may be recovered frequently from the digestive tract. ibv infec-tion of the proventriculus was firstly recorded in china [ ] then detected with unam- ibv mexican variant that produced decrease in the proventricular gland papillary branching and electrodense material scattered in proventriculus with a structure consistent with coronaviruses [ ] . to the best of our knowledge, this is the first time that the prototype ibv strain m was also detected within the muscle layer of the proventriculus. because ibv causes an upper respiratory tract disease, viral antigens in nasal mucosa, trachea and lung were expected. ibv m viral antigen was found in the renal tubules and glomeruli. this observation is consistent with the finding that ibv m has also been isolated and/or detected in kidneys of naturally or experimentally inoculated chickens immunohistochemical detection of ibv antigens in chicken embryos after experimental infection with ibv m figure immunohistochemical detection of ibv antigens in chicken embryos after experimental infection with ibv m . (a) proventriculus, focally extensive, there is strong immunolabelling of smooth muscle cells and scattered cells within the vasculature (arrows). bar = μm (b) brain, within the cytoplasm of numerous neurons there is strong staining of ibv antigen. bar = μm (c) spleen, scattered through the parenchyma there are numerous singular polygonal cells with red intracytoplasmic staining for ibv antigen, interpreted to be macrophages. bar = μm (d) myocardium, capillary endothelium stains strongly positive for ibv antigen. bar = μm. [ , , ] . as earlier described for other ibv genotypes, antigens of m were present not only in renal tubules, but also in the glomerular tuft epithelium [ ] . the detection of ibv antigen in the spleen, is discussed controversially in the literature. in some reports, ibv antigens or mrna were not found within the spleen [ ] [ ] [ ] , in others, splenic infection was observed [ , , ] . studies to determine virus distribution in embryonic tissues were conducted previously [ , ] indicating that ibv antigen or nucleic acid was present in trachea, bursa, kidney, intestine, lung, heart, esophagus, mesentery, shell gland, and air sac, but not in spleen or thymus. the different virus distribution between the present paper and other papers may be due to the difference of embryo age at inoculation; days old embryos (present paper) and or days old embryos [ , ] . it is probable that more immature tissues are more susceptible for ibv. the susceptibility for ibv infection is associated with the expression of , linked sialic acid which is used by the virus for primary attachment to the cell surface [ , ] . for tight binding and subsequent fusion with the cellular membrane interaction with a second receptor appears to be required [ ] . the initial target of avian influenza viruses and ibv in chicken is the respiratory epithelium. presence of , -linked sialic acid is the prerequisite for avian influenza viruses to initiate respiratory infection. this molecule may also be used by ibv for infection of the respiratory tract. ibv, like avian influenza viruses, infects many non-respiratory tissues, including alimentary tract, oviduct and kidney [ , ] . the broad distribution of , -linked sialic acid in different organs and species contradicts the view that this type of sugar is a major determinant of the narrow host tropism of ibv. in this study ibv antigen was found in musculature of both gizzard and proventriculus of inoculated embryos which is consistent with the known presence of , -linked sialic acid receptors in the intestinal tract of chicken [ ] . thus it raises the question whether ibv infection of the proventriculus is a classic feature of ibv and occurs in association with an old ibv strain. a recently isolated m strain [ ] resulted in an increased proventriculus index - days after experimental infection of -day-old chickens [mohamed aa: studies on infectious proventricultis in broiler chicken. master thesis, in progress]. for our knowledge, ibv was neither isolated from nor detected in the brain of young or adult chickens [ ] . our finding that ibv antigens are present in the nervous system of embryonic chicken, may be explained by the presence of polysialylated n-cam (neural cell adhesion molecules) in chicken embryos which might mediate virus entry into the neurons. the abundance of polysialylated n-cam declines gradually during the embryonic development and the synthesis dramatically decreases right before birth [ , ] . our results show that the classic ibv strain m exhibits a wide tissue tropism including the nervous system and the proventriculus in chicken embryos and demonstrate that ihc as described here is a very sensitive tool for detection of productive virus replication in situ and therefore allows further studies to improve the understanding of the pathogenesis of the ibv infection. the ibv strain m used in the current study was kindly provided by m. hess, intervet, boxmeer, nl. chicken embryo kidney cells (cek) were prepared from specific pathogen free (spf) embryonated chicken eggs (ece) as described elsewhere [ ] . briefly, kidneys from -dayold spf ece were isolated, washed with hanks' balanced salts solution, minced and disaggregated in trypsin solution. after centrifugation the cells were resuspended in dulbeccos' modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) and grown at °c in a % co incubator to confluent monolayers. twelve ten days old spf ece were inoculated in the allantoic sac with μl of ibv strain m ( eid ). inoculated ece were candled twice daily. at , , or h dead and/or living whole embryos as well as allantoic fluid and cam were collected. embryos died at any time after inoculation, were also sampled accordingly. the allantoic fluid of inoculated eggs was harvested and clarified by centrifugation for min at g. the obtained supernatants were used for titration in cek. the presence of ibv antigens was investigated in cam and embryos using the avidin-biotin complex (abc) method for immunohistochemistry [ ] . to this end, sampled cam and individual whole embryos, containing all organs were fixed for h in carnoy solution (absolute ethyl alcohol:chloroform:acetic acid; : : ) followed by dehydration in absolute ethanol, and paraffin waxembedding. sections were cut at μm, and mounted on electrostatically charged glass slides. tissue sections (cam and whole embryos cut in transversal or sagittal orientation) were dewaxed in xylene, rehydrated, treated with % hydrogen peroxide and then subjected to antigen retrieval by microwaving in mm citric buffer (ph . ) for min. treated slides were cooled at room temperature for min. non specific background staining was blocked by incubating the sections for min. with goat serum. the sections were then incubated for h with : rabbit anti-ibv polyclonal serum (prepared previously in the laboratory of g. keil, fli, using purified ibv m ) in tris buffered saline (tbs), % bovine serum albumin (bsa), in each tissue randomly selected areas of each compartment were evaluated at high power by light microscopy. the judgments were made semiquantitatively via side-byside comparisons of one section to another and the purpose was to evaluate antigen distribution in relation to labelling intensity in different embryonic tissues. μl of tenfold serial dilutions in mem were added to cek cells per well. after h of incubation, cells were fixed with acetone-methanol, and virus titers expressed as tissue culture infective dose fifty (tcid ) were determined by indirect immunofluorescence using a polyclonal serum raised in rabbits against purified ibv m virions. ibv infected cek cells were fixed with acetone-methanol for min. after washing, cells were incubated with the polyclonal rabbit anti-ibv serum ( : ) for h and subsequently with fitc-conjugated goat anti-rabbit antibodies (sigma) ( : ) for h. both primary and secondary antibodies were diluted in pbs. plates were rinsed three times with pbs after each step. infectious bronchitis coronaviruses in poultry and other birds characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens infectious bronchitis sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent the use of chicken tracheal organ cultures for isolation and assay of avian bronchitis comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohistochemistry histopathology and immunohistochemistry of renal lesions due to infectious bronchitis virus in chicks detection of avian infectious bronchitis using in situ hybridization and recombinant dna advances in avian diagnostic technology. xth wld vet poult assoc congress american association of avian pathologists differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction-fragment-length polymorphism analysis identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s- gene detection of infectious bronchitis virus genetic differences among chicken embryos in response to inoculation with an isolate of infectious bronchitis virus use of allantoic cells for the detection of avian infectious bronchitis virus comparative growth kinetic studies on avian infectious bronchitis virus in different systems infectious bronchitis virus replication in the chicken embryo related cell line a monoclonal antibody-based immunoperoxidase procedure for rapid detection of infectious bronchitis virus in infected tissues avian infectious bronchitis virus distribution in tissues of chicks the pathogenesis of nephritis in chicks induced by infectious bronchitis virus tissue tropism of three cloacal isolates and massachusetts strain of infectious bronchitis virus gastric gross and microscopic lesions caused by the unam- variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos nephrosis in laying chickens caused by massachusetts-type infectious bronchitis virus s gene sequence analysis of a nephro-pathogenic strain of avian infectious bronchitis virus in egypt in vitro characterization and pathogenesis of egypt/beni-suef/ ; a novel genotype of infectious bronchitis virus early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe the control of avian infectious bronchitis/nephrosis in australia establishment of persistent avian infectious bronchitis virus infection in antibodyfree and antibody-positive chickens recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china the quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza a viruses to human type receptors properties and developmental regulation of polysialyltransferase activity in the chicken embryo brain developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, pst and stx cell culture methods iontophoretic application of unconjugated cholera toxin b subunit (ctb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons we thank katrin giesow and gabriele czerwinski for excellent technical assistance. the authors declare that they have no competing interests. asa performed virus inoculation in ece and cell culture, ifa, contributed to ihc, analyzed the data, and drafted the manuscript. pz contributed to ihc and histopathological examination, jv performed virus isolation and ifa. gmk and jpt initiated the project, provide continued directions and critically reviewed the manuscript. jpt also performed and supervised histopathology and ihc. all authors read and approved the final manuscript. key: cord- -r chpw y authors: wu, xinwei; hong, hua; yue, jinya; wu, yejian; li, xiangzhong; jiang, liyun; li, lei; li, qiaoyan; gao, guoquan; yang, xia title: inhibitory effect of small interfering rna on dengue virus replication in mosquito cells date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: r chpw y background: dengue viruses (dens) are the wildest transmitted mosquito-borne pathogens throughout tropical and sub-tropical regions worldwide. infection with dens can cause severe flu-like illness and potentially fatal hemorrhagic fever. although rna interference triggered by long-length dsrna was considered a potent antiviral pathway in the mosquito, only limited studies of the value of small interfering rna (sirna) have been conducted. results: a nt sirna targeting the membrane glycoprotein precursor gene of den- was synthesized and transfected into mosquito c / cells followed by challenge with den. the stability of the sirna in cells was monitored by flow cytometry. the antiviral effect of sirna was evaluated by measurement of cell survival rate using the mtt method and viral rna was quantitated with real-time rt-pcr. the presence of cells containing sirna at . , , , , days after transfection were . %, . %, . %, . % and . %, respectively. after days incubation with den, there was reduced cytopathic effect, increased cell survival rate ( . ± . % vs . ± . %) and reduced viral rna copies (ct value . ± . vs . ± . ) detected in transfected c / cells. conclusions: our data showed that synthetic sirna against the den- membrane glycoprotein precursor gene effectively inhibited den- viral rna replication and increased c / cell survival rate. sirna may offer a potential new strategy for prevention and treatment of den infection. dengue viruses (dens) are the wildest transmitted arbovirus members of the family flaviviridae, genus flavivirus, and compose four serotypes, den- , , , and . as the etiologic agents, dens can cause severe flu-like illness called dengue fever (df), and sometimes lethal complication called dengue haemorrhagic fever (dhf) and dengue shock syndrome (dss) [ , ] . they transmit diseases to human beings primarily through mosquitoes, mainly aedes aegypti and aedes albopictus. with dramatically growth in recent decades, df affects million people and results in about , deaths annually, mostly in tropical and sub-tropical regions. dhf has become a leading cause of serious illness and death among children in some asian countries [ ] . unfortunately, effective vaccines or therapies against the infection are still not available [ ] . rna interference (rnai) is a sequence-specific rna degradation process in the cytoplasm of eukaryotic cells triggered by double-stranded rna (dsrna), widely existing in many species from nematode to human [ ] [ ] [ ] [ ] . upon introduction into the cells, exogenous dsrnas are cut into - nt small interfering rna (sirna) by an rnase iii-like enzyme called dicer. the sirnas form rna-induced silencing complex (risc) with other cellular components, and lead to the cleavage of their homologous transcript and eventually the silencing of specific gene [ ] [ ] [ ] . rnai is believed to be an effective endogenous mechanism for host cells to defense against virus attack [ ] , and has been applied as an exogenous measure to inhibit viral replication, such as hiv [ , ] , influenza a virus [ ] , hbv [ ] and sars-cov [ ] . den is one of the first animal viruses that could be efficiently inhibited by rnai [ , ] . like other flaviviruses, den generates intracellular dsrna as an intermediate of their replication, which may induce rnai in the host cells. a new explanation for mosquitoes' non-pathogenic and persistent infections of den is that rnai could be an important modulator [ ] . exogenous long length dsrna corresponding to den sequences, introduced by either plasmid or sindbis viruses, has been proven to mediate rnai in mosquito c / cells and lead to inhibition of den replication in cultured mosquito cells [ , ] . genetically modified aedes aegypti has been raised to develop dengue virus resistance [ ] [ ] [ ] . the mixtures of den specific small interfering rnas, the hallmark of rnai, were detected in all aforementioned studies. but little was known about the role of single sirna with particular target sequence in the inhibition of den replication. our present study was designed to investigate if a single sirna has the inhibitory effect on den- replication in mosquito cells. effects of sirna on c / survival to den challenge cpe in each group was observed at the th dpi. the virus positive control group and control sirna group showed a large number of cpe up to ++++, characterized by cell swelling and fusing, and reduced cell number; while normal group and sirna group showed no cpe and no observable decrease in cell number. the cell survival rate of c / cells, measured by mtt assay, of sirna group was . % ± . %, control-sirna group was . % ± . %, and virus positive control group was . % ± . %. compared with virus positive control group, the cell survival rate of sirna group increased by . -fold (n = , p < . ), but sirna control group showed no significant difference (n = , p > . ) ( figure ). the amount of intracellular den- viral loading was detected by real time rt-pcr at the th dpi. the ct value of den- rna of sirna group was . ± . , control-sirna group was . ± . , virus-positive control group was . ± . , and no viral rna detectable in normal control group. compared with the virus positive control group, sirna group showed significant decrease of viral loading (n = , p < . ), but control sirna group showed no significant difference (n = , p > . ) (table ). the viral rna amount of sirna group was reduced for . fold (about fold), the inhibition rate was . %. after transfection with fam-labeled sirna, fluorescent signals can be detected in more than half of c / cells cytoplasm under a fluorescent microscope. during the incubation, the fluorescent intensity in the cells gradually decreased ( figure ). and the percentage of fam positive cell at h, days , , , and were . %, . %, . %, . %, and . %, respectively, by flow cytometry (figure ). as a rapid developing technology, rnai has not only become a powerful tool for studying gene function and development of gene-based therapies, but also been widely used in anti-virus researches. precious studies suggested that, at the molecular, cellular and individual levels, rnai can potentially be used to block viral transmission and thus prevent the viral diseases [ , ] . with the high efficiency, specificity and low cytotoxicity, rnai offered a new promise of anti-viral therapy. for ssrna viruses such as den, there genomes are exposed within cytoplasm and become potential targets for rnai. this likely happened at the moment between the uncoating of viral rna and the viral replication [ ] . virus resistance has been proven to be generated by rnai triggered from exogenous den-specific dsrna. adelman et al. [ ] showed this resistance by a nt dsrna homologous to the type ii dengue virus prm gene which was expressed by plasmid, in cultured mosquito c / cells. travanty et al. [ ] developed transgenic mosquito lines that transcribed the same nt dsrna with insect promoters, but failed to express in critical site for den replication such as midguts. franz et al. [ ] increased the size of dsrna to nt and succeeded in midgut expression and viral transmission diminishing. in addition to den- , alternation of the replication kinetics of den- has also proven to be triggered by dsrna in c / cells [ ] . however, long dsrna fragments are associated with higher cost in synthesis, poorer stability, and raise the chance for mismatch rate. at the same time, introduction of dsrnas longer than base pairs into mammalian cells will activate the potent interferon and protein kinase r antiviral pathways, resulting in non-sequencespecific effects that can include apoptosis [ ] . therefore, these disadvantages largely restrict the use of long dsrna in pre-clinical and clinical applications. sirnas are degraded products of dsrna with - nt in length, and have no such disadvantages because of their short length. they are also described as "hallmark" of rnai in all previously published papers. we hypotheses that the sirna derived from the same fraction can have the same inhibitory effect as the dsrna did. therefore, in this study, we designed and synthesized nt sirnas against the den-i viral prm gene, and investigated their inhibition effects of dengue virus replication in transfected c / cells. in four sirnas we designed, only one showed the function to reduce cpe after den infection. as expected, the location of this sirna in den- is inside the corresponding location of nt dsrna reported before in den- . with transfection of the selected sirna, c / cells showed reduced cpe, and increased cell survival by . folds and eliminated viral rna by about . % compared to virus infection only group, at the th dpi. these data indicate that sirna against den viral genome can effectively inhibit viral rna replication in the c / cells, protect host cell from viral attack, suggesting its potential role in prevention and treatment of dengue fever. recent study have shown [ ] that aedes aegypti can produce dsrnas homologous to dengue viral genes and trigger an intrinsic sirna anti-viral action, but this endogenous anti-viral mechanism can not effectively inhibit the replication of dengue virus. data presented in our study revealed that exogenous sirna in aedes albopictus cells is effective in inhibiting viral dna replication. therefore, it is possible that the anti-viral mechanisms mediated by exogenous and endogenous sirnas may act synergistically in the protection of cells from viral attacks, although this hypothesis needs further researches to prove. because the short sirnas are unstable inside the cells, and also diluted by continuous cell division, the sirna contents in the cells decline over time resulting in the weakening of interference effect. from the data in this study, sirnas can be successfully transfected into % of the cells but this percentage gradually reduced with time and sirna molecules only retained in . % of the cells at the seventh day post transfection. therefore, maintaining the continuity of rna interference is crucial for the promotion and application of rnai technology. alternative form of vectors, such as retrovirus [ ] and nano device [ ] , will be applied in the future. since currently there are no effective therapies or vaccines against dengue fever, the use of rnai to suppress dengue virus replication and protect cells from viral attacks will undutiful provide a new research strategy for the prevention and treatment of this disease. our data showed that synthetic sirna against the den membrane glycoprotein precursor gene effectively inhibited den viral rna replication and increased c / cell survival rate. sirna may offer a potential new strategy for prevention and treatment of den infection. the stability and inhibitory efficiency of sirna need further improvement in the future. type den strain gz - and aedes albopictus c / cell line were both from the department of virolo-gy&immunology in guangzhou center for disease control and prevention. c / cells were grown at °c, in eagle's minimal essential medium (mem, gibco, usa) supplemented with % fetal bovine serum (fbs, gibco, usa), μg/ml penicillin and streptomycin, ph . (maintain medium). cells were passaged every to days to maintain exponential growth. den- strain gz - was passaged by infecting monolayers of c / cells and viruses were harvested at - days. tcid of virus was measured for viral challenge. four pairs of sirnas against different parts den- viral genome were designed online (qiagen, german) based on the common sequences of the epidemic strain gz - in guangzhou city of china in (genbank access number ef ), den- reference virus strain (genbank access number eu ), and other den strains. four sirna fragments (densi- ~ ) were synthesized and purified by page electrophoresis (ambion, usa). sirnas was also labeled with fam and purified for visualization after transfection (ambion, usa). a sirna fragment inconsistent containing sequence with den was used as the negative control (qiagen, german). c / cells were cultured in maintain medium at °c, and then inoculated in well plates at × /well in . ml medium the day before transfection. when % % cells grew into monolayer, they were transfected with sirna molecules according to the manual for hiperfect transfection reagent kit (qiagen, german). . μg sirna and μl hiperfect transfection reagent were used for each well. after cultured at °c for hours, the medium with transfection reagent was removed. cells were washed by mem, challenged by tcid den- and cultured at °c in maintain medium for further investigation. the cytopathic effect (cpe) of den- infection on c / cells, including cell rounding, syncytium formation and cell death, was evaluated under a light microscope, and scored based on the severity from (no cpe observed, no cell death) to the most severe level ++++ (cpe observed in % cells). at the th dpi, mtt assay was applied to measure cell viability. μl mtt was added to every well and incubated for hours then replaced with ml dmso. the -well plate was shaken at °c for minutes and od of every well was obtained in a microplate reader. the od value obtained from the normal control group was set as % viability, and the cell survival rate of other groups was calculated as their respective od value divided by that of the control group. . real-time rt-pcr detection of dengue virus rna in c / cells at the th dpi, c / cells were collected and viral rna was isolated with qiaamp viral rna extraction kit (qiagen, german), and quantified with the dengue virus real-time fluorescent rt-pcr detection kit (shenzhen taitai genomics, china). pcr reaction conditions were: one cycle of °c for min and °c for min, then cycles of °c for sec and °c for sec. the relative amount of viral load in each group was represented by ct value, and the changes between groups were calculated by comparative ct (Δct) values. c / cells were transfected with fam-labeled sirnas and cultured for days. fluorescence signal was observed under fluorescence microscope at hours, , , , and days. cells were harvested at every time point, and measured by flow cytometry. sirna positive cell rate was calculated as the percentage of cells containing fluorescent signals. all values were presented as mean ± s.d. statistical significance was evaluated using the two-tailed mann-whitney u-test; p < . was considered significant. who: world health organization: dengue and dengue haemorrhagic fever. fact sheet no. global spread and persistence of dengue dengue virus-mosquito interactions prospects for a dengue virus vaccine potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rna as a target of double-stranded rna mediated genetic interference in caenorhabditis elegans rna interference is mediated by -and -nucleotide rnas rnai in human cells: basic structural and functional features of small interfering rna an rna-directed nuclease mediates post transcriptional gene silencing in drosophila cells mechanisms of gene silencing by double-stranded rna revealing the world of rna interference inhibition of virus replication by rna interference sustained small interfering rna-mediated human immunodeficiency virus type inhibition in primary macrophages lentiviral delivery of short hairpin rnas protects cd t cells from multiple clades and primary isolates of hiv small interfering rna targeting m gene induces effective and long term inhibition of influenza a virus replication inhibition of hepatitis b virus replication by small interference rna induces expression of mica in hepg . . cells inhibition of genes expression of sars coronavirus by synthetic small interfering rnas rna silencing, arthropod-borne virus and mosquitoes dengue virus type infections of aedes aegypti are modulated by the mosquito's rna interference pathway rna silencing of dengue virus type replication in transformed c / mosquito cells transcribing an inverted-repeat rna derived from the virus genome sindbis virus-induced silencing of dengue viruses in mosquitoes developing arbovirus resistance in mosquitoes using rna interference to develop dengue virus resistance in genetically modified aedes aegypti engineering rna interference-based resistance to dengue virus type in genetically modified aedes aegypti architecture of the flaviviral replication complex inhibition of viral gene expression and replication in mosquito cells by dsrna-triggered rna interference antisense rna function and fate of duplex rna in cells of higher eukaryotes octaarginine-modified multifunctional envelope-type nano device for sirna effective suppression of dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution authors' contributions wx and hh performed majority of the experiments and wrote the part of material and methods. yj, wy and lx performed sirna transfection experiments. jl, ll and lq cooperated on cell and virus cultures. yx and gg designed the experiments and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -kgbvld y authors: houspie, lieselot; de coster, sarah; keyaerts, els; narongsack, phouthalack; de roy, rikka; talboom, ive; sisk, maura; maes, piet; verbeeck, jannick; van ranst, marc title: exhaled breath condensate sampling is not a new method for detection of respiratory viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: kgbvld y background: exhaled breath condensate (ebc) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. methods: in our study, volunteers experiencing upper airway infection were recruited over the winter and early spring of / and the first half of the winter of / . ninety-nine ebcs were successfully obtained and screened for commonly circulating respiratory viruses. to investigate the efficiency of virus isolation from ebc, a nasal swab was taken in parallel from a subset of volunteers. the combined use of the ecovent device with the rtube™ allowed the registration of the exhaled volume and breathing frequency during collection. in this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. results: viral screening resulted in the detection of different viruses in ebc and/or nasal swabs: rhinovirus, human respiratory syncytial virus b, influenza a and influenza b. rhinovirus was detected in ebcs and ebc was influenza b positive. we report a viral detection rate of % for the ebcs, which is much lower than the detection rate of . % observed using nasal swabs. conclusion: although very promising, ebc collection using the rtube™ is not reliable for diagnosis of respiratory infections. human respiratory tract infections represent the most commonly encountered infections worldwide. in the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (ns), bronchoalveolar lavages (bal), nasopharyngeal aspirates and sputum samples. the acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. therefore, exhaled breath condensate (ebc) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (copd), asthma, cystic fibrosis, lung cancer etc. ebcs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [ , ] . this observation has created a growing interest in the use of ebc as a new sampling method for the screening of respiratory viruses infecting the upper airways. at first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. however, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. recently, the bronchiole fluid film burst model has been described [ ] . this model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. the aim of this study was to investigate whether the ebc collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. therefore we screened the ebc samples with virus specific pcr assays targeting in this study, ebcs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in table ), using a commercially available condenser (rtube™, respiratory research inc., charlottesville, virginia, usa). the patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for minutes. no nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the t-shaped section of the mouthpiece. in a first part of the study that started during the winter and spring of / , ebc samples were collected from patients who voluntary presented themselves to our laboratory. the majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the catholic university of leuven. the samples were collected with the aluminium cooler sleeve chilled at - °c. in the fall and first half of the winter of / , condensates were collected from patients who presented themselves to their general practitioner. due to practical circumstances, the condensates were collected with the cooler chilled at - °c. for out of collections, the rtube™ was connected by a custom made connectingpiece to the ecovent (jaeger, germany). this device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. additionally, a ns was obtained in parallel with the condensate collection from each patient. all ebcs were immediately stored at - °c. nasal swabs (ns) were refrigerated. after viral dna and rna extraction, ebc samples and nasal swabs were stored at - °c. three specimens were excluded from the study due to incorrect condensate collection. a short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. this study was approved by the medical ethics committee of the university hospital of leuven and informed consents were received from all participants. viral dna and rna were isolated with the qiaamp minelute virus kit (qiagen, westburg, the netherlands) according to the instruction manual. ebc extracts were eluted in μl elution buffer and ns extracts in μl elution buffer. the breath condensates were screened for respiratory rna viruses (cov nl , e and oc , rv, hmpv, infa&b and piv - ) [ ] [ ] [ ] [ ] using a onestep rt-pcr kit (qiagen, westburg, the netherlands) in a μl reaction containing μl of the extracted rna, . μm of forward and reverse primers (table ), . μl one step enzyme mix, μl × one step rt-pcr buffer and μm of each dntp. for adenovirus screening, a dna pcr was carried out for which the amplification reaction mix contained . μm forward primer (adfw) and reverse primer (adrv), . mm dntps, μl buffer c and u taq polymerase in a final volume of μl. the pcr primers used were located in conserved regions of the genomes of the respiratory pathogens ( table ). the reactions were carried out in a t thermocycler (westburg, leusden, the netherlands) with an initial reverse transcription step for rna viruses at °c for min, followed by pcr activation at °c for s, cycles of amplification followed by a final extension step for min at °c. the dna amplification program was initiated with a denaturation step at °c for min, followed by cycles of °c for s, °c for s and a final extension step at °c for min. the amplicons were subjected to a % polyacrylamide gel and visualised under uv light by staining with ethidium bromide. pcr products were purified using the invitek msb spin pcrapace kit and cycle sequenced in forward and reverse direction using the abi prism big-dye termination cycle sequencing ready reaction kit (applied biosystems, foster city, ca, usa). sequence analysis was performed with the abi genetic analyser (applied biosystems, foster city, ca, usa). consensus sequences were obtained using the seqman ii software (dnastar, madison, wis.). for samples from hrsv was detected using a rt-pcr assay as previously described [ , ] . in brief, a multiplex mix was prepared in a final volume of μl using μl extracted rna, . μl of eurogentec one-step reverse transcriptase qpcr master mix containing rox as a passive reference, . μl euroscript + rt & rnase inhibitor (eurogentec, seraing, belgium) nm of hrsv-a and -b specific forward and reverse primers and nm of hrsv-a and -b mgb probes. crna standards were constructed using the megashortscript t kit (ambion, austin, tx, usa) and spectrophotometrically quantified. the viral load of rv positive samples were quantified by qrt-pcr as described in the manuscript published by lu and coworkers [ ] . the eurogentec one-step reverse transcriptase qpcr kit was used for preparation of the master mix as described above. the primerset hrsv-af f - ctgtgatagarttccaacaaaagaaca [ , ] hrsv-af f - agttacacctgcattaacactaaattcc [ , ] hrsv-bn n - ggctccagaatataggcatgattc [ , ] hrsv-bn n - tggttattacaagaagagcagctatacacagt [ , ] mgb probes and probe, located in 'utr, were added to a final concentration of μm and . μm, respectively. crna standards were constructed based on the pcr product of sample using the megascript kit (ambion, austin, tx, usa). quantification was performed with a spectrophotometer at nm and converted to the molecule number [ ] . tenfold serial dilutions, allowing detection in a range of . × to . × rna copies were used. the rt-pcr assays were carried out on a abi prism sequence detection system (applied biosystems, foster city, ca, usa). an initial reverse transcription step was performed at °c for min, followed by a denaturation step at °c for min. finally, an amplification step of cycli at °c for sec and min at °c was completed. ( . %) men, with a median age of (range - years). age and gender was missing for participants of the second group. in total, % of the participants were between - years old. only % were younger than years old and % were older than years. in totality, patients ( . %) were already feeling ill for to days at the day the sample was obtained. seven volunteers ( . %) were symptomatic for to days and participants ( . %) were already ill for more than days at the day of sample collection. data on the duration of symptoms was lacking for patients. almost all volunteers experienced at least symptoms except for two patients (table ) . forty-seven ( . %) volunteers complained about a constant runny or stuffy nose, ( . %) had frequent sneezing events and ( . %) participants had a serious sore throat (table ) . in a first part of the study, we collected ebcs. screening of the ebcs for respiratory viruses (table ) , showed rv ( . %) positive samples (table ). in a second part, we collected ebcs from patients that presented themselves to their general practitioner. two of these ebcs were positive for one of the investigated respiratory viruses, for rv and for infb. to inspect the detection rate of respiratory viruses in the condensate, a ns was taken from this second group of volunteers for comparison. in out of ns ( . %), one or more viral pathogens were isolated. viral screening of the ns resulted in the detection of rv, infa (subtype h n ) and hrsv-b. quantification of the hrsv-b viral load demonstrated for samples and viral titers of . × rna copies/ml and . × rna copies/ml respectively. the rv rt-pcr assay did not allow the quantification of all samples that tested positive for rv by pcr ( table ) . presence of the same pathogen in both the ebc and the ns was confirmed for only sample: sample , which tested positive for rv in both the ebc and the ns. for sample , rv was detected in the ns and analysis of the ebc demonstrated an infb infection. for ebc samples that were collected in the fall and winter of / , measurements with the ecovent in (table , sample ) was positive for infb when using the rtube™ in combination with the ecovent. in theory, the viral generation rate (number of viral rna copies exhaled per minute) can be predicted by quantification of the exhaled viral load. then, an estimation of the rna copies per litre exhaled air or per minute can be calculated. quantification of the exhaled infb would allow us to predict the generation rate for this virus. due to insufficient sample volume, we could not determine the number of rna copies in the sample. collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. the collection of ebc is easy to perform and can be conducted in a home environment. this method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, bal, aspirates, etc. this aspect renders the method very attractive for routine laboratory diagnostics of viral infections. most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable teflon filter on which exhaled particles impact [ ] [ ] [ ] . with the rtube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. until now, this is the study with the largest subset of volunteers that investigated ebc as a specimen for the detection of respiratory viruses. previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. the study performed by fabian and colleagues, included volunteers [ ] . huynh and co-workers recruited volunteers for exhaled breath sampling [ ] . in the study by stelzer-braid et al., ebcs were analysed [ ] and st-george et al. report the participation of adults [ ] . these studies have focused on the detection of infa and -b, piv - , hrsv and hmpv, while we have screened the samples for a panel of commonly circulating respiratory viruses. based on the analysis of ebcs ( ebcs were excluded), our results support the exhalation of rv and infb in % of our samples. since many of the volunteers had already been experiencing symptoms for to days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. for common cold infections it is suggested that a person may already be infectious for or days before experiencing any symptoms. however, in a second part of our study we started collecting ebcs in parallel with nasal swabs from patients presenting themselves to their medical doctor, to days after onset of symptoms. only for condensate the same pathogen was detected in both the ebc and the ns. the detection rate for respiratory viral pathogens in the ns was . % which is much higher than the % detection rate in the ebcs. the low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. the discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. patients that delivered a positive ns may have possibly suffered from an upper airway infection whereas ebc positive volunteers may have experienced a more advanced, lower respiratory tract infection. however, the effect of nasal inhalation on ebc collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. patients with positive ebc samples were experiencing symptoms for maximum two days at the time of collection. however, this was not different for patients with positive ns. six patients that provided positive ns were experiencing symptoms for a longer period at the time of collection (table ). in the group of volunteers that provided an ebc negative or ebc and ns negative sample, the manifestation of symptoms were reported ranging from day to more than two weeks. when reported symptoms were compared between ebc positive patients ( ) and ns positive patients ( ) , % and % in the positive ns group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the ebc positive group. in all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. volunteers were not diagnosed with other pathogens before participation in the study. since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative ns are positive for bacteria or other pathogens causing respiratory illness. recently, one study reported a detection rate of % for influenza in ebc [ ] . this is in the same range of the detection rate that we report for respiratory viruses in general. other studies with a limited number of patients, describe a markedly higher sensitivity of to % [ ] [ ] [ ] but the higher percentage may be due to the low number of participants subjects were included [ ] . remarkably, the studies reporting this higher detection rate used collections masks, while the study using the rtube™ reported comparable findings. face masks consist of electret which trap viruses based on permanently charged fibres [ ] . in addition, the teflon filter has μm pores which will retain all larger particles. possibly, the lower detection rate can partly be explained by the fact that the rtube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. the qrt-pcr developed by lu and coworkers for the detection of rv, did not allow the assessment of the viral load present in the ebc samples [ ] . also for ns, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. for diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in ebc since it is unpredictable how diluted the viral particles in the specimen are. recently, nested qrt-pcr assays have been developed to allow a more sensitive detection of viruses in aerosols [ ] . also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. the participants that were recruited in the study of fabian and coworkers were years of age and older [ ] . for hospitalized children a much higher rate of virus positive samples is reported [ ] . in our study, the majority of volunteers were between and years old. only two children less than years and elderly people (> years) were included. one of the children tested positive for infa in the ns, but the infection was not confirmed in the ebc. for influenza, an exhaled generation rate of < . to influenza rna copies per minute was predicted by quantifying the virus aerosols that impacted on a removable teflon filter of a collection mask [ ] . we used the rtube™ in combination with the ecovent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. in this way, when the number of rna copies in the ebc is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. unfortunately, we were not able to predict a virus generation rate for infb since viral load remained undetermined. although an inventive, new and promising method, ebc collected by the rtube™ does not appear to be appropriate for diagnosis of respiratory infections. nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. this technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly rv. in addition, ebc collection from patients during respiratory infections may be further investigated for biomarker patterns. in calves that were experimentally infected with bovine rsv, an increase in leukotriene b , indicating oxidative stress, was observed. this increased level was also associated with the development of bronchial hyperresponsiveness [ ] . in humans, a transiently elevated h o level was observed during common cold infection. this marker returned to baseline values when volunteers recovered from infection. h o has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [ ] . in infa infected volunteers, an increased co level was observed during upper respiratory infection. this observation might imply that co is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [ ] . therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. this may contribute to the biomarker profiles established for diseases like asthma and copd, for which viral infections are suggested to trigger or exacerbate symptoms [ ] . exhaled breath condensate: methodological recommendations and unresolved questions analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications the mechanism of breath aerosol formation simultaneous detection and identification of human parainfluenza viruses , , and from clinical samples by multiplex pcr a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium 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influenza viruses with nucleic acid-based methods a nested real-time pcr assay has an increased sensitivity suitable for detection of viruses in aerosol studies evaluation of the measurement of leukotriene b concentrations in exhaled condensate as a noninvasive method for assessing mediators of inflammation in the lungs of calves hydrogen peroxide in breath condensate during a common cold increased carbon monoxide in exhaled air of subjects with upper respiratory tract infections exacerbations of asthma and chronic obstructive pulmonary disease (copd): focus on virus induced exacerbations development and evaluation of nuclisens basic kit nasba for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection rapid typing of human adenoviruses by a general pcr combined with restriction endonuclease analysis assay for ' noncoding region analysis of all human rhinovirus prototype strains submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank the volunteers for their cooperation and interest in our study. this research was supported in part by a grant of the institute for the promotion of innovation by science and technology in flanders (iwt) as part of "simid", project number , and by the european community under the epiwork grant (ec-ict contract ). piet maes is supported by a postdoctoral grant from the 'fonds voor wetenschappelijk onderzoek (fwo)-vlaanderen'. we thank our colleagues of the laboratory of clinical virology for helpful comments and critical reading of the manuscript. the authors declare that they have no competing interests. authors' contributions lh designed, executed and coordinated the study. sdc contributed in the sample acquirement and laboratory analysis. ek, pm and jv participated in the execution of the study and added helpful suggestions during preparation of the manuscript. pn cooperated in this study and participated in the recruitment of volunteers and sample collections in a clinical setting. rdr, it, ms work as medical doctors at the medical center of the university leuven and supported our study by recruiting volunteers for our study. all author have read and approved the manuscript. key: cord- -qzm wgde authors: ellermann-eriksen, svend title: macrophages and cytokines in the early defence against herpes simplex virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qzm wgde herpes simplex virus (hsv) type and are old viruses, with a history of evolution shared with humans. thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. in rare situations, however, the primary infection becomes generalized or involves the brain. normally, the primary hsv infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. an early and light struggle inhibiting some hsv replication will spare the host from the real war against huge amounts of virus later in infection. as far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a hsv infection are discussed in this review. generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. in a first wave of responses, cytokines, primarily type i interferons (ifn) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. in the next wave, interleukin (il)- together with the above and other cytokines induce production of ifn-γ in mainly nk cells. many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. this results in the generation of an alliance against the viral enemy. however, these heavy weapons have to be controlled to avoid too much harm to the host. by il- and others, these reactions are hampered, but they are still allowed in foci of hsv replication, thus focusing the activity to only relevant sites. so, no hero does it alone. rather, an alliance of cytokines, macrophages and other cells seems to play a central role. implications of this for future treatment modalities are shortly considered. virus-host interactions are crucial for the outcome of infections. several strategies have been utilized by viruses to overcome the host defence. for the virus to be successful, these evasive strategies have to be balanced with the pathology induced and the possibilities of transmission to new susceptible individuals. the mammalian host utilizes ubiquitous and redundant antiviral defence mechanisms. in different viral infections, different parts of the host defence seem to be crucial. however, the redundancy ensures that other systems are ready to take over, if one of them fails. the final outcome of a viral infection depends on a delicate regulation and timing of these antiviral effector mechanisms in response to the invading virus. a viral infection of an individual thus involves a conflict between the virus and the host, which could conceptually be viewed upon as a human controversy escalating to invasion and armed struggle. to understand the resulting course of events it is important to know each party of the conflict and to conduct an analysis of the powerful weapons held by each of the combatants. the present review analyzes the early non-specific events in the conflict upon herpes simplex virus (hsv) infection. initially, each participant of the conflict, the infecting hsv and the non-specific antiviral weapons of the host, are described. subsequently, the early events of the conflict, the armament, early strikes and the opening battle between hsv and the host are discussed. insight into the early non-specific defence mechanisms are important for our understanding of the conflict and may indicate how to intervene during serious systemic infections. herpesviruses are ubiquitous viruses generally infecting humans early in life. the majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. the initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. the viruses are phylogenetically old and humans and herpesviruses have evolved together [ ] . this co-evolution has created viruses which are well adapted to the human host and environment. thus, herpesviruses are capable of coping with the human immune defence in a balanced manner generally without serious threads to the life of the host. infection with a foreign herpesvirus, normally hosted by another species, does not always hold this balance, and the pathology is unpredictable. this is seen when humans are infected with the simian b virus, which often shows serious clinical outcome [ ] . the human herpes simplex viruses were initially identified by lowenstein, who passed it onto rabbits in , and found it to be sensitive to alcohol and higher temperatures [ ] . the viruses were classified into two serologically different types by schneweiss in [ ] , and these are now known to belong to the subfamily of alphaherpes-virinae together with varicella-zoster virus. these alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. the two types of herpes simplex virus confer the genera simplexvirus and - , which were formally designated by the international committee on taxonomy of viruses as human herpesvirus (hhv) and [ ] . herpes simplex virus (hsv) type and type are very closely related, showing a homology at the dna level of % in protein coding regions and less in noncoding regions [ ] . the genetic map of the two herpes simplex viruses is colinear [ ] , and the genomes are of approximately the same size, hsv- of kbp [ ] and hsv- of kbp, and code for corresponding genes [ ] . the minor sequence variations give different cleavage sites for restriction endonucleases, which has been used intensively as an important epidemiological tool [ ] [ ] [ ] . as all other herpesviruses the herpes simplex viruses are enveloped, icosahedral dna viruses with a capsid of approximately nm ( fig. ) [ ] . the envelope holds at least different glycoproteins protruding from the outer side (gb, gc, gd, ge, gg, gh, gi, gk, gl, and gm). the glycoproteins are primarily responsible for attachment to cellular receptors and fusion of membranes (especially gb and gd) [ ] [ ] [ ] [ ] . in addition, there are two unglycosylated proteins in the viral envelope. the glycoproteins of the envelope have several immunoregulatory effects besides their primary more mechanical functions in viral attachment and entry [ ] [ ] [ ] [ ] [ ] . in the space between the envelope and the capsid, the complete viral particles posses an almost amorphous structure which was termed the tegument by roizman and furlong [ ] . the tegument consists of several viral proteins involved in the initial phases of viral infection and replication such as transport of the viral dna out of the capsid [ ] , early shutoff of cellular protein synthesis (vhs) [ ] , and initiation of transcription of viral genes (α-trans-inducing factors) [ ] . besides the tegument seen in complete viral particles, tegument-like structures are seen in enveloped particles lacking a capsid and dna, the so called light particles [ , ] . the capsid is composed of a complex icosahedral structure of capsomeres, each with a central channel running from the outside to the interior of the capsid. inside the capsid the double stranded linear dna is packed as a spool with the ends in close proximity [ , , ] . the genome consists of a long (l) and a short (s) segment which are covalently linked [ ] , and contains a high density of genetic information with about open reading frames (orf) and encodes approximately polypeptides [ , ] , of which only are required for replication of the virus in cultured cells [ , ] . the viral genes are expressed in a cascade in groups classified as immediate early (ie, α), early (β), early late (γ ) and late (γ ) genes, each with a certain characteristic group of promoters regulating the sequential expression [ , ] . generally, the α-gene products are transcription inducers, the β-gene products are viral enzymes such as the thymidine kinase and the viral dna polymerase, and the products of γgenes are the structural proteins of the viral particle [ ] . the viral transcriptional chain is closed by some of the tegument proteins (e.g. vp /vmw ) which are γ-gene products with structural properties in the tegument of the viral particle and besides this harbour transcriptioninducing capacity upon α-gene promoters crucial in the induction of the next replication cycle of the virus [ , ] . the hsv infection is initiated by adsorption of the viral particle via gb or gc to a cellular receptor, which is a heparan sulphate chain on cellular proteoglycans [ ] . thus hsv adsorption can be inhibited by heparin and soluble heparan sulfates [ , ] . this initial binding, in which gc is important but dispensable, is of greater significance for hsv- than for hsv- , a divergence which could have implications for the different pathogenic patterns of the two strains [ , ] . furthermore, trapping of hsv to heparan sulfate motives in the tissues, e.g. basal laminas, may be of importance for containment of the infection at a specific site [ ] . binding to the heparan sulfate-containing cellular receptors, which are in size with the hsv particle itself, is reversible, and serves to concentrate the viral particle in near proximity to the cell ( fig. ) [ , , ] . a crucial step is then conducted by gd binding to an entry receptor, of which three classes has been described [ ] . these include herpes virus entry mediator (hvem), later designated as herpes virus entry protein a (hvea), which is a member of the tumour necrosis factor receptor family, nectin- (hvec) and nectin- (hveb), both members of the immunoglobulin superfamily, and heparan sulfate sites modified by -o-sulfotransferases [ ] [ ] [ ] [ ] . the differential use of these receptors is of importance for hsv entry of different cell types and infection of polarized cells [ ] [ ] [ ] [ ] [ ] , exemplified by nectin- , which is of importance in infection of the vaginal mucosa [ ] . upon binding to one of these entry receptors, conformational changes in gd lead to interaction with gb or gh-gl dimer, which results in membrane fusion by a mechanism not known in detail ( fig. ) [ , ] . the membrane fusion can take place both with the plasma membrane on the surface of the target cell and with an endosomal membrane after intraluminal ph-reduction, as it is seen for some other enveloped viruses [ , - ]. following these initial steps of infection several immunomodulatory cellular events are induced, but the potential importance of signalling through receptors involved in adsorption and membrane fusion is only scarcely analysed [ ] . the receptor molecule hvem is by its normal ligand capable of inducing activation of nuclear factor κb (nf-κb) and activation of t cells. by interaction with hsv-gd these receptor responses are inhibited. thus, the hsv interaction with at least one of its receptors has multiple potentials for modulation of the host response to the infection [ , ] . upon fusion, the hsv nucleocapsid is transported by microtubules to a nuclear membrane pore where the viral dna is released into the nucleus [ , ]. both viral tegument products and cellular kinases are responsible for the initiation of α-gene transcription [ ] . in these initial events the determination of whether it will lead to a lytic infection cycle or a latent infection seems to be directed largely by the infected cell type in question [ , ] . a key event in this seems to be early induction of latency-associated transcripts (lats) with sequences antisense to the infected cell protein null (icp ) and icp [ ] [ ] [ ] . in the hsv composition and entry figure hsv composition and entry. electron micrograph of negatively stained hsv particle with indications of major structural elements. important mediators of adsorption to cells ( ), receptor binding ( ) and fusion of membranes ( ) during the process of infection are drawn stylistically. initial phase of the lytic replication cycle, the ie-gene products, besides being transcription factors for the next wave of viral proteins, intimately regulate cellular functions in favour of viral replication and immune evasion [ , ] . of these, the icp , a promiscuous transactivator without much dna-binding capacity, forces the cell to a pre-dividing state optimal for viral protein synthesis [ , ] . furthermore, icp is active in inhibiting immune mechanisms such as interferon production and antiviral effects of interferons [ - ] and induces degradation of cellular proteins, involving the proteasome [ , ]. very early in infection, the first transcriptional activity is seen just inside the nuclear membrane at the site were the viral dna enters the nucleus [ ] . the produced icp colocalizes with the promyelocytic leukaemia (pml) nuclear bodies and initiates degradation of these, an event which seems to be important for productive replication of the virus [ , ] . icp binds to parental viral dna which is juxta-localized to the pml bodies, and later, when the bodies are degraded, replication compartments are formed, in which also icp can be found [ , , ] . icp affects the posttranscriptional polyadenylation and splicing of rna, and it is thus an element of the delayed host protein shutoff [ ] . immune evasion is additionally induced by the ie protein icp which binds to transporter associated with antigen processing, tap /tap and blocks the presentation of viral peptides by the major histocompatibility complex (mhc)-i [ ]. the hsv progeny is formed in the nucleus of the infected cell, where the viral dna is packed into preformed capsids. these are assembled with the tegument proteins and bud through the inner nuclear membrane to the perinuclear space [ ] . the route of virus from here to the external side of the cell is controversial. apparently two routes of viral egress are possible [ ] . one way is by continuous passage through vesicles and the golgi apparatus, where the membrane proteins are modified. the other route is by fusion of the newly acquired envelope with the outer nuclear membrane or the membrane of a vesicle, generating naked nucleocapsids in the cytoplasm. from here a new budding event should take place, for instance into the golgi apparatus. the progeny virus thus acquires the envelope from other membranes, than the inner nuclear lamella, as it is indicated by analysis of membrane lipids [ ] . increasing evidence is pointing at this latter possibility of de-envelopment and re-envelopment as the dominating route of hsv egress [ - ]. the progress of hsv infection in tissues is influenced by the capacity of hsv to infect adjacent cells directly through cell junctions. the virus is thus avoiding exposure to extracellular substances such as antibodies and complement. the glycoproteins ge and gi are crucial for this kind of polarised transmission which primarily takes place in epithelial infections [ , ] . as it is the case at the molecular level, the two herpes simplex viruses show similarities in their clinical appearance, both giving rise to primary infections of mucosal membranes and showing latency in sensory nerve ganglia [ ] . the primary infections with hsv are often asymptomatic, especially at young age, but in a minority of cases vesicular or ulcerative lesions are seen. although hsv- and - can give rise to indistinguishable clinical infections, there are differences in the anatomical distribution of these infections, as described in by dowdle et al. [ ] . hsv- is predominantly giving rise to infections above the waist, and hsv- to infections below the waist. this pattern is, however, not as straightforward as primarily described. in the last decades changes in both prevalence and distribution of hsv infections have been seen. the overall prevalence of hsv infection is very different in different countries and ethnic and social populations [ ] [ ] [ ] . a decline in hsv prevalence has been observed in the western countries, probably because of improved socioeconomic conditions [ ] [ ] [ ] . in parallel to the decline in prevalence, the aetiology of herpes genitalis has changed in several countries, presumably because of altered human habits and conditions of life [ ] . in some areas of the world the proportion of genital infections caused by hsv- is still low ( - %) the aetiology of a genital infection is not insignificant, in that the frequency of recurrence is higher in hsv type infected individuals than in those infected with type [ , ] . the frequency of primary and recurrent infections with both hsv- and - has been reported to be higher among women than men [ , , ] . overall, these epidemiological changes could have implications for the risk of neonatal infection from vaginal delivery, in that more women are seronegative at delivery and thus a higher number have the risk of caching a primary hsv infection. on the other hand, less hsv is circulating, reducing the risk of those who are susceptible. clinical appearance and pathogenesis as described above, primary infection with hsv is most often asymptomatic, especially in younger children [ ] . however, some individuals experience a symptomatic primary infection with vesicular herpetic gingivostomatitis or in adolescence more often a pharyngitis [ ] . as it is the case with orofacial infections, a primary genital hsv infection can be both asymptomatic and symptomatic with ulcerative lesions and with or without generalized symptoms such as fever, headache etc. [ , ] . rarely, the infection disseminates to one or several organs giving rise to infections such as necrotising hepatitis, meningitis, encephalitis or to disseminated intravascular coagulopathy [ ] [ ] [ ] [ ] . such a clinical course, although uncommon, is most often seen in immunosuppressed patients e.g. transplant patients, neonates or pregnant women [ ] [ ] [ ] . in pregnancy, primary infection with hsv without previous seroconversion at the time of delivery seems to be the main risk factor for infection of the newborn [ , ] . genital hsv reactivations at labour only seem to posses a minor risk for neonatal infection of the baby [ , ] , but in spite of this, approximately % of neonates infected are born by asymptomatic women [ ] . the amount of virus in vaginal secretions during reactivations is much lower than the amount of virus in primary infections, and in reactivated cases maternal antibodies furthermore seems to be protective for the neonate [ , [ ] [ ] [ ] [ ] . when transmitted, the course of hsv infection in the newborn varies. in the pre-acyclovir era about one third of cases were mucocutaneus infections only involving the skin, mouth and eyes, one third were infections of the central nervous system (cns) with or without mucocutaneus involvement, and the last third were disseminated infections involving multiple organs, including the liver, lungs, adrenals, and often the cns [ ] . of these, neonates with a generalized infection had a one-year mortality of approximately %, those with cns-infections had intermediary mortality, and nearly no mortality was seen in the group of patients with only mucocutaneus involvement [ ] . in infected with multi-organ involvement the deaths are often set off by infection of liver or lungs or by coagulopathy. sequelae, such as mental or neurological disabilities are seen in some of those with cns involvement [ ] . now a day, after initiation of high-dose acyclovir treatment, the mortality and sequela rates have dropped [ ] . the clinical pattern of neonatal hsv infections has changed in that less of the mucocutaneus infections disseminate to generalized infections when treated [ ] . even with high-dose acyclovir, improvements in treatment protocols are still needed, because the mortality is still as high as % in disseminated infections. reduction in the time from debut of symptoms to initiation of therapy is vital and passive immunotherapy with hsv-specific antibodies could posses a potential as adjuvant to the antiviral treatment [ , , ] . other adjuvant treatment modalities are still needed in both neonatal infections and in generalized infections at later ages. the pathology of hsv infections is mainly caused by a direct cytopathic effect of the virus, resulting in cellular lysis and focal necrosis of the infected area [ , , ] . in tissues capable of regeneration, this is not devastating, provided that the lesions do not totally destroy the organ or result in functional disability during the infection. in the brain, however, the capacity for regeneration is small, and larger necroses induced by viral infection will result in life-long sequelae [ , ] . a delicate balance exists between the direct hsv-induced pathology and the immunopathology induced by immune reactions to the virus and the toxic and functional side effects of these reactions [ ] . immunopathogenesis seems to be the main aspect of hsv stromal keratitis, which often leads to blindness [ , ] . the scarification from this infection has even been attributed to autoimmunity by molecular mimicry [ ] . weak immune response to the virus leads to severe infections because of massive viral replication and dissemination. an immense immune reaction, especially with high amounts of virus to trigger a response, can bring about increased symptoms of infection, local symptoms such as high intra-cerebral pressure or pulmonary complications, as well as generalized or septic symptoms [ , [ ] [ ] [ ] [ ] . it is thus clear that early control of hsv replication in the initial phases of infection is crucial for the host. early containment or at least inhibition of viral replication can prevent dissemination of the infection, and the early nonspecific immune reactions thus have the potential to inhibit development of a symptomatic infection. obviously the host will benefit from an attenuated or asymptomatic course of infection, but hsv -with the potential of subsequent reactivation from a latent site -could also benefit from such a course of infection, in that the host will survive and the activity of the host in society will not be hampered by symptoms from infection. thus, the hsv has excellent chances to reach new susceptible hosts which bring the virus and the host in a situation of mutual benefit [ ] . with phagocytic activity. in metchnikoff named them macrophages (large eaters) in contrast to microphages (the polymorphonuclear leukocytes) [ ] , and in aschoff defined the reticuloendothelial system by the criteria of uptake of vital dye [ ] . the macrophages are now more precisely defined as an important member of the mononuclear phagocyte system, defined in by van furth and colleagues [ ] . in the tissues they constitute a dynamic pool of cells with many functional capabilities, among which the capacity of phagocytosis, microbial killing, motility, and adherence to surfaces are classic [ ] . the macrophages originate from the bone marrow, where proliferating promonocytes give rise to monocytes which enter the blood stream [ ] . after a mean circulation time of approximately / day, the blood monocytes migrate to the tissues [ ] . in the tissues the monocytes differentiate into macrophages with characteristics determined by the environment of the tissue in question [ ] . the tissue macrophages in the major organs are represented by kupffer cells in the liver, alveolar and interstitial lung macrophages, spleenic and sinusoidal lymph node macrophages, microglia in the brain, osteoclasts in bone, and langerhans cells of the skin. thus, macrophages are strategically situated all over the body taking care of debris from the organism itself and foreign material, among others invading microorganisms, including viruses [ , ] . macrophages in different organs have different characteristics and functional capabilities and can not totally substitute one another in studies on macrophages [ , [ ] [ ] [ ] [ ] . likewise, macrophages from different species can possess differences in their functional capability, e.g. the capacity for nitric oxide (no) production [ , ] . macrophages in tissues are, as described above, in part originating directly from monocytes, but they are also in part originating from local proliferation. this local proliferation in the tissues is performed by newly recruited monocytes, and in the steady state situation they only constitute a small fraction of the mononuclear phagocytes present [ ] . of the monocytes produced in the bone marrow of mice and passing through the blood, approximately half are targeting the liver, % are going to the lungs, % to the spleen and % to the peritoneal cavity [ ] [ ] [ ] . in the lungs, % of tissue macrophages in the steady-state originate from monocyte influx and % from local proliferation [ ] . this proportion might vary between different tissues, as the lifespan of tissue macrophages in different organs also varies from around days in mouse spleen to approximately one month for alveolar macrophages [ , ] . in the skin, langerhans cells are a very stable and long-lived population of cells staying there for at least month in the steady-state situation. however, in inflammation the langerhans cells are within weeks replaced and supplemented by circulating mononuclear cells [ ] . when an inflammatory process is initiated, the dynamics of monocytes and macrophages are changed. monocytes and other white blood cells are produced and recruited from the bone marrow, and the white blood cell count in the circulation is increased. the monocytes are mainly passing through the blood to become tissue macrophages, and the number of macrophages in the inflamed tissue can be increased by more than ten times [ ] . in inflamed tissue the local proliferation of macrophages does not seem to increase, although the number of newly recruited cells is high, indicating that the differentiation of monocytes in the tissues is accelerated [ ] . the differentiation of monocytes and activation of macrophages have been a focus of interest for many years because of the observation that macrophage activation is crucial in the defence against many intracellular pathogens [ ] [ ] [ ] [ ] . it became clear relatively early that lymphocytes and soluble factors secreted by these (lymphokines) are important in activation of macrophages for killing of intracellular bacteria, e.g. listeria [ ] . in the killing of bacteria, interferon (ifn)-γ was shown to be an important stimulator of macrophage activation [ ] . as mechanisms in performance of the killing simple toxic substances of reactive oxygen species (ros) and nitric oxide were identified and seem to conduct their action in synergy [ ] [ ] [ ] . the toxic substances are chemically simple, but their production and regulation in macrophages are very complex and still a matter of intense studies [ ] . the state of the activated macrophage has changed conceptually from being viewed as one specific condition of the cell towards a more dynamic picture, provoked by the fact that macrophages activated by different means show different phenotypical characteristics [ , ] . the activated macrophage is now viewed as a cell with floating characteristics of many functional capacities regulated by a multitude of stimulating substances, such as the cytokine environment, hormones, and pathogenic and foreign substances [ , ] . among variables, controlling macrophage activity in infected individuals, are the genetic constitutions of the host. the genetic background has been shown to be of importance for the regulation of both basic proliferation and function of macrophages and for the more specific antimicrobial responses [ , ] . soluble mediators of lymphocyte activities were described as early as , but the first lymphokines/cytokines found and characterized were the type i interferons. soon after, many other soluble mediators of lymphocyte and monocyte/macrophage activities were found [ ] [ ] [ ] . the term lymphokine was introduced by dumonde et al. in , to describe lymphocyte derived factors, and the term monokine was used as a description of factors coming from the mononuclear phagocyte system, both acting on many cells, primarily leukocytes [ ] . because of a broader view on origin and function of these factors, the term cytokine is now more often used. each cytokine was originally named according to biological activity in a functional assay, which often gave several different names to one cytokine, and thus confusion at the molecular level. to straighten this out, a numerical nomenclature of interleukins (between leukocytes) was introduced in [ ] . this numbering system has clarified the field, but since it has no mnemonic functional anchorage it has drawn critique since then [ ] [ ] [ ] . the cytokines are generally smaller proteins, some composed of two subunits, utilizing specific receptors on target cells for induction of their functional effects. they are structurally related in three families, with the prototypes being il- , il- and il- [ ] . functionally, cytokines are highly potent regulatory proteins acting in a paracrine or autocrine manner at picomolar concentrations [ ] . the cytokine receptors are also structurally clustered in families, and functionally utilize a battery of overlapping kinases and nuclear binding proteins in their signalling pathway and thus have overlapping functions [ ] . the final functional capacity of the effector cell thus reflects the cytokine environment experienced by the cell [ ] . thus the cytokines comprise a network of factors inducing or inhibiting each others secretion and function in different cells, giving rise to a constantly floating landscape of a large array of functional capacities [ ] . in the early hours of a viral infection, the cytokines produced by cells infected or coming into contact with viral products are vital in conduction of the innate immune response to the infection [ , ] . the interferons (ifns) were described and named in by isaacs and lindenmann [ ] , who characterized the substances involved in the previously described interference of one virus with the replication of another unrelated virus, and the interfering activity of inactivated influenza virus with the subsequent infection of chorioallantoic membranes [ ] [ ] [ ] . the ifns were the first cytokines described in detail, and thus provided the fundamental basis for the understanding of the cytokine concept [ ] . the ifns are divided into three major groups. the two original groups of ifns are designated type i and type ii, type i being the so called non-immune ifn, and type ii the immune ifn. type ii (ifn-γ) is produced in high amounts as part of a specific immune reaction, whereas the type i ifns can be produced by many cell types in response to, in immunological terms, non-spe-cific stimulation. the many functions of ifns and the growing understanding of signalling and regulation indicate that ifn analogues may play a major role in the next generation of new antiviral compounds [ ] . the type i ifns are a diverse group of cytokines, consisting of ifn-α, ifn-β, ifn-ε, ifn-κ, ifn-ω, ifn-δ, ifn-τ, and ifn-ξ/limitin [ , ] . the first five of these are expressed in humans, and their relative production depends on the stimulus and the cell type in question. the ifn-α family consists of multiple species and some of these in different allelic forms in both humans and mice. in humans ifn-α genes and one pseudogene and in mice ifn-α genes and pseudogenes have been identified, clustering on chromosome in mouse and chromosome in man [ ] . the functional importance of such a diversity is largely unknown. the subtypes differ in potency and have previously been shown to vary in their profile of activities [ , ] , but new studies show correlation between antiproliferative and antiviral effects of various ifn-α species [ ] . thus, it seems that the importance of the diversity could come from varying expression patterns of the different ifn-α species. most of the α ifns are n-glycosylated, but glycosylation does not correlate with activity of the molecule, but rather with in vivo stability, and recombinant ifns are shown to have activity comparable with that of the naturally produced molecules [ , ] . only one ifn-β species exists, coded by a gene situated in the ifn type i cluster on chromosome in mouse and chromosome in man, as described above [ ] . the natural ifn-α and -β have a molecular weight of - kda and most species retain stability at ph [ ] . all type i ifns bind to one common receptor composed of two subunits, ifn-α-receptor(r) and ifn-αr . the ifnα/β receptor (ifnar) signal through the jak/stat-pathway by phosphorylation of the janus kinase (jak) , tyrosine kinase (tyk) , signal transducer and activator of transcription (stat) and stat , and induces genes with an ifn-stimulated response element (isre) in their promoter [ , ] . generally the type i ifns exhibit a huge range of biological effects, such as antiviral and antiproliferative effects, stimulation of immune cells such as t cells, natural killer (nk) cells, monocytes, macrophages, and dendritic cells, increased expression of mhc-i, activation of pro-apoptotic genes and inhibition of anti-apoptotic mechanisms, modulation of cellular differentiation, and inhibition of angiogenesis [ ] . the newly discovered ifn-ξ/limitin also interacts with the ifn-α/β receptor, and is regarded as a type i ifn [ , ] . antiviral activity of ifn-ξ has been shown against many viruses including hsv, and it exhibits both immunomodulatory and anti-tumour effects, but the lymphosuppressive activity is less than that of ifn-α [ , ] . a human homolog of ifn-ξ could thus have interesting potential in the therapy of tumours and viral infections. the type ii ifn is represented by only one member, the ifn-γ [ ] . structurally, ifn-γ is distinct from the type i ifns, and it signals through a different receptor. for many years ifn-γ was thought only to be expressed by t cells. later the large granular lymphocytes (nk cells) were recognised as important producers by the fact that ia-antigen (mhc-ii) expression on mouse macrophages could be induced by listeria monocytogenes infection in scid mice lacking t cells [ ] [ ] [ ] . in recent years it has, however, been clear that other cell types, originally thought not to be producers of ifn-γ, are in fact capable of ifn-γ expression. so now macrophages, b cells, nkt cells and professional antigen-presenting cells are also recognized as ifnγ producers in certain situations [ ] [ ] [ ] [ ] [ ] [ ] . induction and production of ifn-γ in antigen-presenting cells and nk cells seem to be vital in the early non-specific response to infections and of importance in the linkage to the adaptive specific responses coming up later [ ] [ ] [ ] . the induction of ifn-γ production in non-t cells (e.g. nk cells) is conducted by cytokines, especially il- in synergy with other proinflammatory cytokines, largely produced by mononuclear phagocytes [ , ] . ifn-γ exerts its effects through a distinct class ii cytokine receptor, the ifn-γ receptor (ifngr), composed of two subunits, ifn-γr and ifn-γr . upon binding of a homodimer of ifn-γ to the receptor complex, jak autophosphorylates and then transphosphorylates jak . activated jak in turn phosphorylates ifn-γr , which allows binding of the stat homodimer to the receptor and subsequent phosphorylation of stat [ ] . the ifngr and stat are preformed as hetero-and homo-dimers, and upon receptor binding, the ifn-γ-ifn-γr -stat complex seems to be internalized and translocated to the nucleus, where the activated stat homodimer binds to dna at gas elements and induces the first wave of responses [ , [ ] [ ] [ ] [ ] [ ] . many of these initial ifn-γ induced products are transcription factors participating in further regulation of the many ifn-induced cellular response. among these products are the ifn regulatory factors (irfs) which stimulate or inhibit transcription of genes possessing an isre in the promoter region [ , ] . for many years the key mediator of macrophage activation during antigen-induced processes was recognised as macrophage activating factor (maf) [ ] . only later, the crucial importance of these effects was attributed to ifn-γ [ , ] . ifn-γ has antiviral activity, but the most important effects of ifn-γ seem to be activation of macro-phages, antigen-presenting cells, and nk cells and inhibition of t-helper type (th ) cells, resulting in a th driven cell-mediated response to infection [ ] . experiments in knock out (ko) mice with deficient ifn-γ, ifngr, or stat expression have shown that this system is of major importance, but not vital, in the host response to viral infections [ ] [ ] [ ] [ ] . besides the two traditional groups of ifns, a new group of ifn-like cytokines has been described in various species and named il- a (ifn-λ ), il- b (ifn-λ ), and il- (ifn-λ ) [ , ] . these cytokines are antiviral proteins interacting with a distinct heterodimeric class ii cytokine receptor composed of ifn-λr and il- r , but sharing with the type i ifns some intracellular signalling pathways through the isre [ ] . thus, they have a largely similar antiviral effect as the type i ifns [ ] . tumour necrosis factor (tnf, former designated tnf-α) and lymphotoxin (lt; former tnf-β) were for many years also known as cachectin from their involvement in cachexia of cancer patients [ ] . tnf is a prototype and the second member of the tnf ligand superfamily (tnfsf ), now encompassing over known signalling molecules, among which the ltα, ltβ, and light (ltlike, exhibits inducible expression, and competes with hsv glycoprotein d for hvem, a receptor expressed by t lymphocytes) are some of the more prominent ligands [ , ] . each member is the ligand of one or two distinct receptors of the tnf receptor family sharing a high degree of homology. the current nomenclature of these ligands and receptors has now been gathered on the internet [ ] . tnf is a type ii transmembrane glycoprotein coded from the human chromosome and from chromosome in mice [ ] . it is synthesized as a kda transmembrane pro-tnf, primarily located in the membranes of the golgi apparatus [ ] . the pro-tnf is cleaved by a metalloprotease releasing the kda extracellular portion of the molecule [ , ] . production and release of tnf from the cell is regulated at both the transcriptional and translational level and by post translational modification as described above [ ] . during hsv infection both preand post-transcriptional regulatory mechanisms are involved in tnf production [ ] . tnf is produced by many cell types of immune origin, primarily mononuclear phagocytes, neutrophils, nk cells and t cells, and has diverse effects on different cells [ ] . both membrane bound and soluble tnf interact as homotrimers with two different receptors, the p tnfr (tnfrsf a) and the p tnfr (tnfrsf b) [ ] . as most other receptors of this family, tnfr holds a death domain important in the pro-apoptotic pathway. tnfr is expressed virtually on every cell type except erythrocytes, whereas tnfr is mostly expressed on endothelial and bone marrow derived cells [ ] . the tnfr activates nf-κb (p , p /rela, and p /relb) by ubiquitin-mediated degradation of inhibitor-κb (iκb) after phosphorylation by an iκb kinase (ikk). besides inducing apoptosis, tnfr also activates nf-κb (p / p ) [ , ] . furthermore, the activator protein (ap- ) is activated by mitogen-activated protein kinases (mapks) and together with nf-κb primarily acts in the proinflammatory pathways. thus, signalling from the tnf receptor family induces a delicate balance between life and death (apoptosis) of the cell. both of the tnf receptors can by proteolytic cleavage be converted to soluble receptors with the capacity to compete with their signalling ancestors, but also act to stabilize the trimeric tnf and thus maintain its activity [ , ] . the tnf superfamily seems to have evolved with the adaptive immune system in vertebrates and is crucial for the embryonic development of lymphoid tissue [ ] . furthermore, tnf is, as a proinflammatory cytokine, involved in activation of many immune cells and is thus an important factor of both the early non-specific and the specific immune response [ ] . the importance of the tnf superfamily in antiviral defence is illustrated by the fact that different viruses have developed mechanisms for interference with nearly every step of activity of this system [ , ] . il- is the prime member of a small group of heterodimeric cytokines, all with the capacity to induce production of ifn-γ in a variety of cells. il- was first described as an nk cell stimulatory factor (nksf) and identified as a heterodimeric molecule composed of a p and a p subunit, which are covalently linked [ ] . the p subunit has homologies to il- , and p is homologous to the extracellular domain of the haematopoietin receptor family, particularly the il- rα chain [ ] . the two il- subunits are coded from different chromosomes, i.e. the human chromosomes and and the mouse chromosomes and , respectively [ ] . these genes are regulated separately, and coordinated induction in the same cell is required for secretion of the biologically active il- p heterodimer [ ] . il- is produced by monocytes, macrophages, dendritic cells, neutrophils and b cells [ , ] . in the initial response of spleen cells in mice injected in vivo with extracts of toxoplasma gondii or with lipopolysaccharide (lps), the cellular source was found to be dendritic cells, but cultured macrophages have by themselves also been shown to produce il- p upon hsv- infection [ , ] . such differences could depend on variations in the signalling mechanisms involved, which is also illustrated by the observation that the production in dendritic cells and macrophages has dif-ferent kinetics. this difference could be brought about by differences in the requirement for co-stimulation with ifn-γ [ ] . a collaborative action of dendritic cells and macrophages could be important, as indicated for il- induction by influenza virus and other inducers [ ] . the receptor for il- is found on nk cells, t cells and dendritic cells and consists of two subunits (β and β ), which signal by the β subunit through the jak/stat pathway, primarily by activated stat [ ] . the primary effect of il- is induction of ifn-γ production in nk cells and t cells, and il- activates the cytotoxic potential of these cells. the ifn-γ locus in nk cells is constitutively demethylated and is thus ready for transcription of the gene, which is in contrast to that of t cells, [ ] . macrophages and nk cells are then stimulated by ifn-γ, resulting in activation for enhanced antimicrobial capacity [ , ] . il- and ifn-γ in conjunction are the main responsible factors for activation of a th -driven adaptive cellular immune response, important for the long-term control of intracellular pathogens [ ] . il- stimulates proliferation of naïve t cells, and in conjunction with ifnγ inhibits th cell differentiation and the production of th cytokines (e.g. il- , il- , and il- ) [ ] . thus il- holds a key position in induction and control of the th response. the il- -induced ifn-γ production is synergistically enhanced by other cytokines such as tnf and il- [ ] , and ifn-γ production can even be induced in macrophages by co-stimulation with il- [ , , ], a cytokine which by itself does not possess major ifn-γinducing capacity [ ] . a positive feed-back loop is initiated by the il- -induced production of ifn-γ, in that ifn-γ is an important primer of il- production, thus accelerating the system [ ]. furthermore, t cells enhance il- production through signals of the proinflammatory tnf family [ ] . in virus-infected macrophages a similar autocrine feed-back loop involving il- , il- , ifn-α/β, and ifn-γ could be speculated [ ] . this potentially harmful situation, with accelerating ifnγ production, regulated in a positive feed-back loop by il- , is inhibited by cytokines possessing anti-inflammatory properties. among these il- holds a crucial position as an inhibitor of il- production, an effect which is also conducted by transforming growth factor-β (tgf-β) [ ] [ ] [ ] .the th cytokines of the other side of the adaptive response, il- and il- , inhibit il- induction in the early phases of stimulation, but later they can be potent inducers of il- production, although they still inhibit many of the ifn-γ-induced activities [ , , ] . phagocytosis of apoptotic cells by macrophages inhibits production of il- , a regulatory mechanism which seems to be important in restriction of the damages induced by uncontrolled defence mechanisms [ ] . injection of high doses of il- to virus-infected mice is toxic, and leads to death with the pathology of tnf-related toxic shock, an effect which was explained by increased sensitivity to the toxic effects of tnf, and found to be dependent on the genetic constitution of the host [ , ] . the small il- cytokine family also includes two other heterodimeric cytokines, il- and il- , and a homodimer of il- p . the latter is found in vivo in mice and functions as an antagonist of il- , but it is debated whether it exists in humans [ , ] . il- is composed of the il- p and a p subunit and likewise binds to a receptor with one of the il- receptor subunits (il- rβ ) and a distinct il- r subunit [ , ] . the production and function of il- is quite similar to that of il- , but il- has a unique capacity to induce proliferation of memory t cells [ ] , and it has been found in nervous ganglia of hsv-infected mice on day of infection [ ] . il- drives il- production of nk cells, which mobilizes neutrophils and promotes production of the proinflammatory cytokines il- , il- , and tnf [ ] . il- is the newest recognized member of the family, constructed of two distinct subunits (ebi and p ), but still with functional capacities alike those of il- [ ] . the functional implications of these later discovered members of the il- family is not yet clear, but it seems as if they are contributors to the overall effects of the il- family and fine-tune the system [ , [ ] [ ] [ ] [ ] . the induction of ifn-γ and activation of nk cells is not only mastered by members of the il- cytokine family. other cytokines, like il- , are also implicated in development, function, and activation of these cells [ , ] . generally, the il- cytokine family has shown itself of importance in early defence against several viral infections, and as a vital inducer and regulator of the adaptive immune response against viruses and other intracellular pathogens [ , , , ] . upon an accelerating pro-inflammatory response induced by initial viral replication the organism has to embank the ifn-γ-activated potentially harmful actions of macrophages and nk cells. important mediators of this embankment are il- and il- , which as described above repress the induction of il- , and thus put a brake on the positive feed-back loop of ifn-γ production [ , ] . furthermore, il- suppresses the production of other proinflammatory cytokines such as tnf and il- [ ] . most importantly, il- and il- are potent inhibitors of the efferent arm of the pro-inflammatory system, and thus inhibit production of reactive oxygen species and nitric oxide. the production of these two potentially harmful effector mechanisms of activated macrophages is hampered by inhibition of production of the responsible enzymes in these reactions, the nadph oxidase and the inducible nitric oxide synthase (inos) [ ] [ ] [ ] . the primary producer cells of il- and il- are the th cells, but these cytokines are also produced by basophils and mast cells [ ] [ ] [ ] . the receptors for il- and il- are expressed on most cells and are composed as dimers of four different chains. il- is the ligand of two receptors: a high-affinity heterodimer of il- rα and the il- r common γ-chain and another heterodimeric receptor composed of il- rα and il- rα . il- binds to three complexes: a high-affinity heterodimer of il- rα and il- rα and two homodimers composed of either il- rα or il- rα , which are both coded from genes on the human x-chromosome [ ] . the immunomodulatory signalling is conducted through the jak/stat-pathway utilizing jak , jak and stat . phosphorylated and homodimerized stat binds to stat binding elements (sbe), which includes gas, and either trans-activates or inhibits transcription of the adjacent genes [ ] . the functions of il- and il- are nearly overlapping with only discreet discrepancies [ , ] . il- was discovered in on the basis of another important effect of the cytokine, namely the ability to induce proliferation of b cells, and it was from this effect in the early years called b cell growth factor [ ] . as this, some other effects of il- are stimulating, in that it furthermore activates other th -like effects such as b cell class-switching and expression of mannose receptor and fc receptor for ige on macrophages [ ] . despite the anti-inflammatory profile il- has in vivo been shown to confer some resistance to hsv infection [ , ] . il- is thus not only an inhibiting cytokine but essentially an immunomodulatory cytokine with regulatory effects on macrophages as well. the early innate defence mechanisms have for many years been regarded as important for the course of many viral infections, including infections with hsv [ ] . the control of viral replication and dissemination during the first days of an hsv infection seems to be vital for the final outcome. if the viral replication is not halted by natural defence mechanisms during induction and maturation of the antigen-specific immune response, the adaptive immune system can be overwhelmed by massive viral infection at the dawn of activity of the specific reactions. the mechanisms of the anti-herpetic natural defence have been analysed extensively. it became relatively early clear that antiviral activity of macrophages [ ] and nk cells [ ] and early activity of the ifn-system [ ] were important mediators of innate resistance to hsv. the relative contribution of each of these players in the early defence has been much debated, and as more interactions and molecular mechanisms are now elucidated, it seems clear that all of these players each hold a crucial position in an integrated antiviral natural defence system. an important model used in the study of resistance mechanisms in defence against generalized infection with hsv is a mouse model, where mice infected intra-peritoneally or intra-venously experience a generalized infection with hsv replication in most organs, including the liver, spleen, and eventually the brain [ ] . the dissemination of infection to the brain and the severity of infection of the peripheral organs depend in part on the age of the mice, as is the case in humans, where neonates have difficulties in controlling a hsv infection [ , [ ] [ ] [ ] . the course of infection in mice also depends on the type of hsv in question. furthermore, in lopez described a differential susceptibility of inbred mice to generalized infection with hsv, and this genetic difference in sensitivity has since been used for analysis of resistance factors of importance for the anti-herpetic defence [ ] . in generalized infections, the genetics of the relative resistance to hsv- was shown to segregate with the x-chromosome [ ] . this pattern of resistance to the generalized infection was for both hsv- and - attributed to a genetically determined difference in the capacity for ifn-α/β production [ , , ] , and it was shown that the x-linked pattern of resistance segregated with the hsv- -induced production of ifn-α/β in macrophages during the first hours of infection [ ] . furthermore, macrophages from female mice respond to hsv with higher ifn-α/β production than macrophages from male mice [ ] . this observation is in line with female mice being more resistant to hsv infection in vivo [ ] . early production of ifn-α/β has been correlated to resistance of hsv infections in several other studies. treatment of mice with antibodies to ifn-α/β increases and accelerates mortality of a generalized hsv- infection and with higher doses of virus, mice are dying already after three to four days, a period where antigen-specific mechanisms are still in the induction and proliferation phase [ ] . furthermore, mice treated with mercuric chloride showed higher titres of hsv- in the first days of infection, an effect which could be correlated to impaired production of ifn-α/β [ , ] . in studies on peripheral hsv infections, such as cutaneous or corneal infections, ifn-α/β has been shown to be produced locally and to restrict the local replication of hsv and infection of nervous ganglia cells of the area, an effect which has also been correlated to the genetic constitution of the host [ ] [ ] [ ] [ ] . the genetic background for the x-linked trait of hsv resistance and ifn-α/β production of macrophages remains unravelled. induction of ifn-α/β upon hsv infection seems to be governed by different mechanisms in different cells [ ] . ifn-α/β can be induced early by both infectious and uv-inactivated hsv in various cells, with the infectious virus being the more potent inducer in mouse peritoneal macrophages, whereas the uv-inactivated virus showed most potency in human peripheral blood mononuclear cells (pbmc) [ , [ ] [ ] [ ] [ ] . production of ifn-α/β was induced by gd of hsv- in pbmc, but not in murine macrophages [ , , ] . in pbmcderived dendritic cells, however, the cellular mannose receptor was shown to be involved [ , ] . furthermore, different toll-like receptors (tlrs) have been shown to react with hsv [ ] . tlrs are transmembrane pattern recognition receptors (prrs) that detect redundant microbial molecular motives and induce antiviral and proinflammatory cytokines in response to alerting signals. in dendritic cells, tlr -signalling, induced by the gc-rich hsv genome, has been shown to govern the induction of ifn-α/β, but tlr -ko mice are still capable of controlling hsv infections in vivo [ ] [ ] [ ] . however, in mouse macrophages the tlrs do not seem to be crucial for ifn-α/β induction upon hsv infection [ ] . this is in agreement with the observation that the majority of ifn-α/β produced by spleen cells and dendritic cells and the total production from bone marrow macrophages was independent of tlr or myd , which is necessary for signalling by most tlrs [ ] . in this study, heat inactivated virus was shown still to induce ifn-α/β in cells utilizing tlr . as resident peritoneal macrophages do not produce ifn-α/β in response to even high doses of heat inactivated hsv, this gives an additional indication of independency from tlr of ifn-α/β production in macrophages [ ] . moreover, efficient induction of ifn-α/β by hsv in macrophages required dsrna-activated protein kinase (pkr) activity and infectivity of the virus [ ] . this is in agreement with the observation that dsrna, which is produced by most viruses during replication, induces ifn through pkr, and not through tlr , which also binds dsrna [ ] . furthermore, another mechanism of ifn induction by dsrna through a rna helicase has been proposed [ ] . the different induction patterns in different cells types, and the fact that ifn-α/β seems largely to be induced by other mechanisms than tlrs, explain the fact that knocking out tlr-signalling by myd did not influence the in vivo infection with hsv in mice [ ] . other tlrs have also been shown to mediate signals in hsv infections. in hsv encephalitis in tlr -ko mice, viral replication seemed unchanged or slightly increased during the first days of infection, and the production of il- and monocyte chemoattractant protein were impaired, but interestingly pathological changes and mortality were reduced [ ] . in relation to the x-linked resistance pattern of hsv infection and ifn production upon hsv infection, it is interesting that some of the tlrs are coded from the xchromosome [ ] . these are the tlr and tlr , which are triggered by guanosine-or uridine-rich ssrna in the endosomal compartment of cells [ , ] . there are, however, no indications that this pathway is implicated in ifn induction in cells during hsv infection, but the question has still not been directly addressed. regulation of the ifn-α/β gene induction is in part governed by activation of the transcription factors irf- and - , which are induced by ifn-α/β itself, resulting in a positive feed back loop, an effect which has been known for years without knowledge of the signalling mechanisms [ ] [ ] [ ] . thus, one possible explanation for the genetic differences in hsv-induced ifn-α/β production could be an elevated physiological level of this ifn self-stimulating system [ ] [ ] [ ] [ ] . an analysis of the levels of irf- and - in normal macrophages from these mice could be of interest. analysis of the levels of the ifn-induced enzyme '- '-oligoadenylate synthetase (oas) in uninfected cells showed low but slightly higher levels in cells from relatively resistant mice [ ] . with lps, cells from the relatively resistant (c bl/ ) mice show an early induction pattern of ifn-α/β, peaking within hours, whereas cells from the susceptible balb/c mice demonstrate a delayed response, peaking hours after induction [ ] . among other transcription factors involved in induction of the various ifn-α/β genes are the heterodimeric nf-κb family, which is activated by tlrs, il- r, and tnfr [ ] . during a hsv infection nf-κb is activated and translocated to the nucleus [ ] . many regulatory mechanisms of nf-κb activation exist, one of them exerted through tnf, which is produced by macrophages very early during hsv infection ( fig. ) [ , , , ] . thus, the responsible mechanisms might be exerted by other regulatory signals, influencing the magnitude of the hsv-triggered ifn-α/β induction pathway, and perhaps not by this pathway in itself [ ] . a number of x-linked immunodeficiencies have been described, one of them being the wiskott-aldrich syndrome with defects in a protein expressed in haematopoietic cells, facilitating reorganization of the actin cytoskeleton, and thus influencing the mobility of immune cells and chemotaxis of macrophages. patients with this x-linked immunodeficiency show aggravated herpetic infections, and cells from some patients seem to produce lower amounts of ifn in response to hsv [ ] [ ] [ ] . cells from patients with another x-linked immunodeficiency with mutations in the cd -ligand, a member of the tnf family, showed decreased ifn-α/β production when infected with hsv- , but these patients apparently show a normal response to viral infections [ , ] . this supports the notion above that other regulatory signals might be involved. the overall effects of the ifn-α/β system, besides the production as described above, are determined by the sensitivity of cells to the secreted ifn-α/β. the effector mechanisms of ifn-α/β on hsv replication are not fully elucidated. several ifn-α/β-activated systems are involved, including the dsrna-activated pkr, which phosphorylates, and thereby inhibits, the elongation initiation factor (eif)- α, resulting in inhibition of translation [ ] . another important mediator of the antiviral activity is the oas system, which activates '- 'oligoadenylate-dependent rnase l with the capacity to degrade single-stranded rna [ ] . lately, the pml bodies have been described as crucial for the anti-hsv effect of ifn-α/ β [ ] . in mice exhibiting a relatively hsv-resistant phenotype, the direct antiviral effect of ifn-α/β in embryonic cells was found to be approximately three-fold higher than in cells from susceptible mice [ ] . data from another study showed comparable results on ifn-α/β sensitivity concerning the replication of encephalomyocarditis virus (emcv) in cells from the same mouse strains [ ] . this phenomenon was inherited as a co-dominant autosomal trait without any apparent influence of x-linked genes [ , ] . further studies in mouse fibroblasts have revealed that tnf intensify the antiviral effect of ifn-α/β and, thus, the in vivo situation seems more complicated ( fig. ) [ , ] . in the original publication on genetics of hsv susceptibility in inbred mice, lopez reported fibroblasts from the different mice to replicate hsv equally, and the same was found in the cells showing differential sensitivity to ifn-α/β [ , ] . in line with these results, the ifn-activated oas, an inhibitor of hsv replication, was induced to a higher degree in cells from the resistant mice upon ifn-α/β treatment [ , , ] . furthermore, the level of stimulated and unstimulated oas was generally found to vary between different inbred mouse strains [ ] . thus, the genetic difference in antiviral action of type i ifns seems to affect the replication of several different viruses and to correlate with resistance to hsv. the viral host protein synthesis shutoff, exerted by the hsv vhs-protein of the tegument, has major effects on the cytokine production of infected cells and reduces the effect of ifn-α/β on hsv replication [ ] . furthermore, the tegument proteins have been shown to induce cellular inhibitors of the jak/stat pathway, resulting in inhibition of both ifn signalling and production [ , ] . the ie protein icp inhibits activation of irf- and thereby also restricts ifn-induced pathways [ - ], and icp , icp and icp induce late shutoff of protein synthesis with decreased mrna stability and thus reduced cytokine production [ , ]. as outlined, it thus seems hsv has evolved several mechanisms to evade the consequences of the ifn-α/β system, which underline the importance of these cytokines in the antiviral defence. during hsv infection macrophages are activated and possess an increased antiviral potential [ , ] . classically, the macrophage antiviral activity has been described as intrinsic or extrinsic [ ] . resting macrophages possess a high degree of intrinsic activity against hsv, generally being non-permissive to viral replication. the macrophages are thus a blind end for the hsv infection, and they can in that way protect other cells from infection, for example as a barrier lining the liver sinusoids [ ] . the extrinsic antiviral activity refers to the ability of macrophages to inactivate virus outside the macrophage itself or to inhibit viral replication in other cells [ ] . the intrinsic antiviral activity depends among other factors on macrophage differentiation and has been correlated to ifn activity, either physiological levels of "spontaneous" preinfection-synthesized or rapidly acting autocrine ifn-α/β [ ] . in that respect, macrophages from mice of the resistant phenotype showed higher intrinsic activity by being less permissive to hsv replication [ , ] . one potential antiviral mechanism of macrophages may be the production of ros. these were originally assigned to bacterial killing, but the effect of ros has also been correlated to antiviral functions, although they might not be of major importance [ ] . the ros are mainly produced by nadph-oxidases (nox), which are membrane-bound multi-component enzymes primarily situated in the phagolysosome [ ] . activation of the nahpd-oxidase, by phosphorylation and fusion of the enzyme subunits, primarily results in production of superoxide anion (o -), which by superoxide dismutase can be converted to hydrogen peroxide (h o ). the h o in turn is then by fe + (fenton reaction) or by fe + and o -(haber-weiss reaction) converted to hydroxyl radical (·oh), hydroxyl anion (oh -) and singlet oxygen ( o ), or by the myeloperoxidase to hypochlorous acid (hocl) [ , ] . small amounts of ros are also produced by the mitochondria and may be of importance as signalling molecules from tnf [ , ] . during hsv infection in vivo, macrophages are activated and achieve an increased capacity to react with a respiratory burst of ros when appropriately triggered, i.e. by phorbol esters (fig. ) [ ] . this macrophage activation is induced early in response to hsv infection, reaching a plateau within the first hours of i.p. infection [ ] . in vitro, macrophages were shown to be the cell type responding with an oxidative burst, and this capacity peaked after only hours of infection with hsv [ ] . this hsv-induced capacity for an increased respiratory burst was shown to be governed by autocrine ifn-α/β as a sine qua non phenomenon [ , ] . nevertheless, tnf was also found to influence the macrophage activation. by itself, tnf reduced the macrophage capacity for a respiratory burst, but in combination with ifn-α/β it synergistically enhanced the ifn-induced activation [ , ] . interestingly, a secreted portion of the hsv-gg acts as a phagocyte chemoattractant and induces production of ros by signalling through the receptor activated by the phorbol esters [ ] . the hsv-induced activation of macrophages in vivo is influenced by the genetic constitution of the host, with the most pronounced activation of macrophages originating from resistant mice, as expected on the basis of the genetics of ifn-α/β production in response to the infection. furthermore, the genetics of the efferent part of the ifn-α/β-mediated hsv-induced activation of macrophages, displayed a co-dominant autosomal trait, as was the case with the antiviral effect of ifn-α/β in fibroblasts [ ] . thus, the genetically-determined sensitivity to ifnα/β seems to be expressed in different cell-types. the influence of tnf on the genetics of this phenomenon has not been addressed. in contrast to these observations, the genetics concerning the antiproliferative effect of ifn-α/β in bone marrow cells seems to be reversed [ , ] . this might, however, be linked, in that ros are shown to activate various signalling molecules, mediate apoptosis, and exhibit antiproliferative effects depending on the dose and time of exposure [ ] . little is known on the potential antiviral effect of ros. by examining peroxidized lipids, which is an oxidative product from ros in tissues, it has been documented that these are produced during the acute hsv infection in vivo, and speculations on antiviral mechanisms have focused on induction of apoptosis [ , , ] . hsv triggers apoptosis of infected cells by several pathways, and the importance of this phenomenon is indicated by the fact that the virus has evolved mechanisms to counteract each of these pathways [ ] . macrophages generally suppress apoptosis in hsv infections, as seen by increased apoptosis in macrophage-depleted mice [ ] . several studies on the mechanisms involved in the early battle against hsv, performed in in vivo animal models, have pointed to ifn-α/β as a crucial player. in adoptive transfer experiments, the effect of adult mouse spleen cells on the initial phase of a generalized hsv infection in suckling mice was conducted by ifn-α/β [ ] . furthermore, administration of a hematopoetic growth factor to neonatal mice increases the number of dendritic cells, b cells and nk cells, and confers resistance in a cutaneous model of hsv infection. the effect in this model could also largely be attributed to the actions of ifn-α/β, with some additional contributions by ifn-γ [ , ] . in ko-mice ifn-α/β was able to control the initial phase of a generalized hsv infection without contributions from nk, t-or b cells, but these latter players were necessary for survival and long term control of the infection [ ] . the importance of an early, local ifn-response in models including in vivo progression and evaluation of final outcome of infection is more unclear, in that many other viral and host factors are of importance in these more complicated models with several stages of infection and involvement of different organs. such models are, however, more close to the normal human hsv infection, starting at an epithelial surface, but to expect that one resistance factor in such a complicated system will come out clear as the responsible factor for the outcome downstream the sequence of events, is too simplistic. nevertheless, induced expression of ifn-α/β in the eye by plasmid dna or an adenovirus vector was shown to inhibit early local replication of hsv and the concomitant spread of virus to the brain and death from encephalitis [ , ] , and in ifn-α/βr ko-mice hsv replicated to much higher titres than in normal mice [ ] . this tells us that the innate and adaptive immune systems exhibit much redundancy, and that ifn-α/β is of vital importance in local inhibition of hsv replication. the multitude of antiviral mechanisms, be it innate or adaptive, have varying effects and importance in the different phases of infection, such as initial local infection, dissemination to other organs, establishment of latency and reactivation, and conclusions can not be drawn from one situation to another. the reactions discussed above, involving production of ifn-α/β and tnf, take place within the first to hours of a hsv infection, and thus are reactions, which can execute an effect within the first replication cycle of the virus. a little later, other cytokines such as il- , il- and ifn-γ are produced and give rise to other weapons in the battle against the virus. they will, in turn, within the next replication cycle execute their actions, with potential harmful consequences for either parts of the conflict. a few hours after the type i ifn and tnf response, macrophages react upon hsv infection with production of il- , which is seen from to hours after infection and on [ , ] . the same was found with other viruses to hours after infection [ ] . in these and other studies, the producers of il- p during viral infection seem to be inflammatory cells, including macrophages, and not the infected stromal cells [ , ] . the il- induction during hsv infection requires infectious virus, and it was shown to be regulated at the transcriptional level [ ] , as it is also the case when it is induced by lps [ , ] . the dependence on infectivity is, however, in conflict with results from in vivo production of il- p and ifn-γ in draining lymph nodes from sites injected with uv-inactivated hsv [ ] . high doses of uv-inactivated virus were used, and some minimal transcription of viral genes could have taken place, although the virus was not replication competent. transcription of the il- p gene in macrophages requires de novo protein synthesis during the inducing hsv infection, which could explain the relatively late appearance of il- production [ , ] . the κb-sequence of the il- p promoter binds nf-κb in hsv-infected cells, and the production of il- p was found to be repressed by an inhibitor of nf-κb activation [ ] . both these observations indicate that signalling through nf-κb is of significance in hsv-induced il- production. in human macrophages, tnf has been shown to inhibit il- p production, but not p production, by a mechanism not involving nf-κb [ ] . furthermore, il- has in a mouse model been shown to stimulate tnf expression [ ] , indicating that tnf can participate in a negative feed-back loop in the regulation of the il- system [ ] . likewise, ifn-α/β has been shown to inhibit il- production in both humans and mice [ ] [ ] [ ] . the implication of such inhibition by ifn-α/β and tnf, which are secreted very early in hsv infections, well before the production of il- , has so far not been elucidated. as described earlier, the il- p induction is influenced by ifn-γ in a positive feed back loop. ifn-γ could activate il- transcription through binding of irf- , - , and - to an isre site in the promoter-region of il- [ , ] . upon hsv infection, ifn-γ is produced as part of the nonspecific response to the virus. a marked synergism between hsv and ifn-γ in il- induction has been demonstrated [ ] , indicating that the il- / ifn-γ autoaccelerating system is of importance during hsv infections. the ifn-γ-inducing activity of the produced il- is pronounced in mouse peritoneal cells after hours of infection with hsv [ ] . in a study by kirchner et al. ifn-γ was detected as early as on day of in vivo hsv infection, and the ifn-γ production was correlated to the genetics of hsv resistance [ ] . during hsv infection, the production of ifn-γ is mainly induced as a concerted action of several factors and not by il- alone. ifn-α/β by itself was shown to be a weak inducer of ifn-γ production by nk cells, but in synergy with il- the production of ifnγ was markedly enhanced [ ] . in elicited peritoneal macrophages, hsv induced efficient ifn-γ production through cooperation of il- , ifn-α/β and il- [ ] . in such a proinflammatory environment even other cells than nk and t cells, e.g. macrophages, might produce lower levels of ifn-γ [ , , ] . il- signals through stat , but stat translocation to the nucleus of nk cells has also been seen after ifn-α/β stimulation [ , ] . likewise, ifn-α/β induces stat phosphorylation in t cells [ ] , indicating that il- and ifn-α/β at this point act through a shared signalling pathway. furthermore, the synergistic action of il- and il- in ifn-γ production by macrophages was shown to be dependent on stat [ ] . in addition to these factors, tnf and il- have also been shown to act in synergy with il- in ifn-γ induction [ , , ] and vice versa, ifn-γ has been shown to synergize with hsv in induction of tnf production [ ] . this further emphasizes the concept of positive feed-back mechanisms in the regulation of early ifn-γ production. the important direct effect of ifn-α/β on hsv replication was found to be enhanced synergistically by ifn-γ in both cell culture and in vivo in mice [ , [ ] [ ] [ ] . this is, however, in conflict with an early study, which could not reveal any synergism between ifn-α/β and ifn-γ on the replication of hsv in human blood mononuclear cells [ ] . synergistic action of the two types of ifn is further supported by the observation of synergism between ifn-γ and tnf on hsv replication in corneal cells, and the fact that this was exerted through production of ifn-β [ ] [ ] [ ] . the effect was, however, greatly dependent on the cell type examined, which could explain the above-mentioned inconsistency. synergism between tnf and ifn-γ in inhibition of hsv replication has now been shown to be mediated by activation of a tryptophan-depleting enzyme [ ] . thus, relatively small amounts of early ifn-γ produced by nk cells in response to il- , ifn-α/β, tnf, and il- could in collaboration with the already present ifn-α/β and tnf have important local effect on hsv replication in permissive cells ( fig. ). this conclusion is further supported by observations in ko mice, indicating that collaborated action of ifn-α/β and ifn-γ is of importance in control of subcutaneous hsv infections [ ] . in vivo studies on hsv infections in immunodeficient, ko, and antibody-treated mice have shown that the il- , second early wave of response figure second early wave of response. regulatory pathways controlling production and action of ifn-γ during early hsv infection. when infected with hsv macrophages (mφ) produce several cytokines, including il- , which stimulate production of ifn-γ, primarily in nk cells. ifn-γ then induces no production in macrophages and stimulate the direct antiviral activity of ifn-α/β in other cells. stimulatory pathways are indicated by green arrows (→), and inhibitory pathways are drawn in red. - / ifn-γ system is able to control the infection, affecting both the survival rate and the hsv titres early in infection [ , ] . the effect of il- in hsv infections seems to be conducted in synergy with il- [ ] , as it has also been shown for vaccinia virus [ ] . in hsv corneal infections in ko mice, il- was shown to participate in the immune pathogenesis [ ] , but in another study utilizing il- encoding plasmid dna, corneal expression of il- reduced the angiogenesis, and thus the pathology of the infection [ ] . however, both studies agreed that il- does not affect the local titres of hsv in the eye. after a thermal injury, wide-spread hsv infections are an important risk, and treatment of injured mice with il- combined with soluble il- r results in augmentation of the ifn-γ production and decreased viral replication and mortality [ ] . in mice infected with murine cytomegalovirus (mcmv) production of il- -induced ifn-γ by nk cells has been demonstrated in vivo, and the system was further shown to lower the viral titres [ , ] . the il- / ifn-γ system seems, however, not to be of importance in all viral infections, in that the latter study could not detect any production of early il- or ifn-γ in a model of infection with the arenavirus lymphocytic choriomeningitis virus. analyses of the il- , - / ifn-γ system in humans with genetic defects and in ko-mice reveal more redundancy in man than in mouse and indicate that the system is of more importance in dna-than in rna-virus infections [ ] . the producers of early ifn-γ, the nk and nkt cells, and the cytokine il- and the transcription factor t-bet, which are both crucial for the differentiation and function of these cells, have all been shown to be decisive for the early control of hsv infection in vivo [ , [ ] [ ] [ ] . although nk cells but not ifn-γ was shown to be decisive for survival from ocular infections [ ] , such an effect of ifn-γ has been seen by others [ ] . furthermore, a review of genetic functional nk cell defects found nk cells and their innate ifn-γ production to be of central importance in herpesvirus infections [ ] . overall, it can be concluded that the il- / ifn-γ system is active in hsv infections and possesses an important antiviral potential, capable of controlling viral replication during the early phases of infection. in macrophages exposed to ifn-γ, the enzyme inducible nitric oxide synthase is induced, which eventually results in production of no from molecular oxygen and a guanidino nitrogen by conversion of l-arginine to l-citrulline [ ] . upon hsv infection, the inos gene is induced, as shown by detection of inos-mrna in infected mouse peritoneal cells and corneal neutrophils [ , ] . the production of no in hsv-infected cultures of resting mouse peritoneal cells, which comprise a mixed population of macrophages, lymphocytes, nk cells etc., is dependent on the virus being infectious [ ] . this is in line with the requirement of infectious hsv for il- production and thus for production of ifn-γ as described previously [ ] . no could itself be involved in a positive feed-back, in that signalling of il- utilizing tyk requires the activity of no [ ] . when exogenous ifn-γ is added to virus-infected cells, a marked synergism is seen. this synergistic effect of hsv on the ifn-γ-induced no production in macrophages was shown to be mediated by autocrine secretion of tnf [ , ] . in line with this, mice with a targeted disruption of the tnf gene showed impaired resistance to hsv and increased viral replication within the first days of infection [ ] , and antibodies to tnf and an inhibitor of no production impaired early control of hsv infection in peripheral nervous tissue [ ] . the induction of inos and the following production of no in response to ifn-γ and hsv is a relatively slow reaction, coming up after about hours of infection [ ] . in in vivo vaginal hsv infections inos mrna could be detected after hours of infection [ ] . thus, the production of this relatively toxic substance is part of the second wave of innate defence mechanisms. the retarded production of no and the requirement for two or more signals for induction of inos are logic considering the in hsv-infected macrophages exposed to ifn-γ, inos is induced synergistically though tnf-induced nf-κb activation and translocation to the nucleus, as shown by binding of a heterodimeric complex of p /p and a homodimer of p to the κb-site of the inos promoter during infection [ ] . the crucial position of nf-κb in the induction of inos and production of no is also indicated by experiments showing that antibodies to tnf inhibit activation of nf-κb and production of no in hsvinfected cells and abolish the synergism between the virus and ifn-γ, an observation which was also seen with inhibitors of nf-κb activation [ ] . further analysis of the signalling mechanism has revealed that the synergism upon hsv infection is influenced by physical interaction of irf- and the nf-κb subunit p and controlled by the isresite and the distal κb-site of the inos promoter ( fig. ) [ ] . a further support for this notion comes from the observation that the dna-binding capacity of nf-κb and the nuclear translocation of irf- have similar kinetics upon hsv infection [ ] and the fact that irf- is essential for inos induction [ ] . induction of other genes such as ifn-β and vascular cell adhesion molecule also involve physical interaction of irf- and nf-κb [ ] , and both irf- and irf- have in other cells types been shown to form complexes with nf-κb [ , ] . another potential mechanism in the synergistic induction of inos could involve complex formation of ifn consensus sequence-binding protein (icsbp or irf- ) and irf- , which is also important for high-output no production but has still not been studied in hsv infections [ ] . thus, high-output no production from activated macrophages is controlled by a "double-lock" signalling mechanism restricting the production of this antiviral toxic substance to sites of active viral replication, and sparing uninfected tissue from the detrimental effects ( fig. ). the antiviral effects of no have been documented in several viral infections, although there clearly exist viruses and conditions where no does not exhibit major antiviral properties. no is thus not a magic bullet against virus infections [ ] . in hsv infections, no has been shown to confer a substantial part of the antiviral activity induced by ifn-γ in a macrophage cell line and to participate in the extrinsic anti-hsv effect of macrophages [ ] [ ] [ ] [ ] . an exogenously added donor of no has in several cell lines been shown to reduce the replication of hsv [ ] . in vivo, analysis of mice treated with an inhibitor of no production showed higher titres of hsv in the lungs but increased survival rates due to reduced inflammation [ ] . recently, a study using another inhibitor of no production has confirmed the anti-herpetic effect of no during a hsv respiratory infection, but in this study mice with inhibited no production showed increased inflammatory responses, symptoms of infection, and mortality ifn-γ il- (at high ifn-γ / stat ) [ ] . replication of hsv during vaginal infection was increased in the presence of an inhibitor of no production, and this enhanced viral replication was most prominent during the first hours of infection [ ] . in inos-ko mice, the herpes virus mcmv replicates to higher titres in various organs and in macrophages, and this results in impaired survival of the animals [ ] . weanling mice with a targeted disruption of the inos gene showed increased hsv replication, but apparently without differences in hsv titres during the first days of infection [ ] , and in adult ko mice, we could not detect any significant effect of no during the early days of a generalised hsv infection (ellermann-eriksen, unpublished results). probably, these in vivo results are due to redundancy of the antiviral system. [ ] . the final effects of no on hsv infections therefore appear to be balanced between antiviral versus toxic effects, and the final outcome seems to depend on the timing, infectious dose, and tissues involved. thus no production in the early phases of hsv infection is one of the effector mechanisms of the innate immune response inhibiting hsv replication, but when overproduced, no might itself result in pathology, as discussed in the following section. as outlined above, positive feed-back mechanisms exist at the afferent side of the early cytokine response, involving especially the production of ifn-γ, il- , ifn-α/β and tnf, and synergisms at the efferent side, resulting in highoutput no production. as a result of coordinated induction of the inos gene by several transcription factors, activated by especially ifn-γ and tnf, a potent early antiviral system is activated. however, no causes damage to dna, proteins and lipids in cells and tissues and could thus be deleterious for the host [ ] [ ] [ ] [ ] . a study in ko-mice indicates that no can be responsible for inflammation and life-threatening symptoms to hsv infection of the lungs [ ] . this effect of no on pulmonary symptoms is also observed in influenza virus infections [ ] , although no inhibits replication of both influenza virus and severe acute respiratory syndrome coronavirus [ , ] . consequently, when this system is activated, it has to be controlled and eventually closed down, as it would otherwise induce unnecessary harm to the host. such negative regulations of the inos gene induction in ifn-γ activated macrophages is conducted by il- and il- [ , , ] . furthermore, tgf-β can exhibit downregulation of no production through several post-transcriptional regulatory mechanisms, but the contribution of these pathways have not been analysed in hsv infections [ , ] . il- production during hsv infection has in vaginal and cns infections been demonstrated on day of infection and to increase for the next days [ , ] . in peritoneal cells from mice infected i.p. pro-duction of il- could be detected at day of infection [ ] . at low ifn-γ concentrations, il- has been shown to inhibit inos induction through stat competition with stat binding to the gas element of the irf- promoter region. this results in reduced expression of the transcription factor irf- , which is crucial for induction of inos [ ] . generally, stat was shown to be a key factor in il- -and il- -induced inhibition of inos gene transcription induced by ifn-γ (fig. ) [ ] . at higher ifn-γ concentrations, activated stat is no longer able to compete with the high amounts of activated stat dimer [ ] . however, in this situation il- is still able to inhibit the production of no from ifn-γ-stimulated macrophages [ , , ] . in the presence of high levels of ifn-γ, il- is not able to alter the induction of irf- , but the production of irf- is increased [ ] . the human promoter region of irf- contains a sbe, and the induction of irf- could thus potentially be mediated by stat binding to this element [ ] . this is in agreement with the fact that irf- is known to compete with the binding of irf- to isre sites and to antagonize the transactivating activity of irf- in the regulation of other ifninduced genes [ ] [ ] [ ] . inhibition of inos expression by high concentrations of irf- relative to irf- has thus been proposed as a controlling mechanism in situations with high levels of ifn-γ ( fig. ) [ ] . furthermore, another mechanism could evolve from the observation that il- signalling can result in disruption of the complex formation of icsbp and irf- and thereby inhibit inos induction [ ] . other mediators of il- -induced repression of inos induction might exist, in that another dnabinding transcriptional repressor competing with irf- has been described [ ] . in ifn-γ activated macrophages the il- -and il- induced inhibition of inos induction can thus be overruled by hsv infection, leading to a sustained no production ( fig. ) [ , ] . this effect of hsv infection is mediated through tnf production and nf-κb activation [ , ] . however, pre-treatment with il- has in a theiler's murine encephalomyelitis virus model showed inhibition of nf-κb activation [ ] . in thioglycollateinduced peritoneal cells, lps and tnf could only overcome the inhibiting effect of il- in situations, where il- was added simultaneously or after the stimulators [ ] , a sequence of events which, however, is in agreement with the sequence of cytokine production in hsv infections. when activated, the nf-κb p physically interacts with irf- and trans-activate inos transcription in hsvinfected and tnf-treated cells [ , ] . it is thus tempting to speculate that the nf-κb-irf- complex has higher affinity for the combined dna-binding site and thus is able to obstruct the binding of irf- to the isre site of the inos promoter and in that way turn the competition towards transcriptional activity ( fig. ) [ ] . this will block the inhibiting effect of il- in foci of hsv replication and open up for no production at sites where the antiviral effect is of more importance than the potential toxicity. in treatment of hsv infections, we have for many years had a very powerful tool in the antiherpetic drug acyclovir and related compounds. but there are still therapeutic problems in the group of patients with generalized or cns infections, and therefore it is tempting and timely to hypothesize on possible future treatment strategies. as described, it is clear that relatively discrete but early actions of the non-specific defence systems are crucial for the long term outcome of the infection. the same holds for the antiviral therapy, and early presumptive therapy and rapid diagnostics could thus potentially improve the final outcome. in the seeking for improved antiviral treatment, adjuvant therapy with anti-hsv antibodies could potentially accelerate the clearance of viral particles, and block viremic dissemination in patients, who are still seronegative at the time of treatment. immunomodulatory treatment modalities imitating the early non-specific antiviral defence, working as described in this review, could be considered. the key players exhibiting the least toxicity by themselves could be used, taking advantage of potential synergy with other cytokines in the foci of hsv infection. in the future, molecules with affinity for various receptors are expected to be produced, and when we know the signalling mechanisms in detail and all the potential interactions, molecular signalling could be addressed directly by pharmaceuticals. in consequence of the crucial position of the type i ifns in innate response to hsv, future analogues of ifn-α/β seem obvious as candidates for adjuvant treatment of severe hsv infections. this could be supplemented with il- , which would give the highest ifn-γ production in foci of hsv infection because of other cytokines such as ifn-α/β, tnf and il- being present there. with focussed production of ifn-γ at sites of active viral replication and treatment with ifn type i analogues the focal antiviral activity could be increased markedly, without too much activity in areas without infection. to hamper systemic consequences of the enhanced proinflammatory reactions, such pro-inflammatory treatment could perhaps benefit from concomitant treatment with il- or other stat -activating therapeutics in the future. this would further focus the activity to sites of active hsv replication. in situations with massive viral replication in nearly all organs, high-dose aciclovir should perhaps only be supplemented with anti-inflammatory medications and inhibitors of tnf, since many of these individuals risk to die from septic reactions. the author(s) declare that they have no competing interests. the family herpesviridae: a brief introduction cercopithecine herpesvirus (b virus) aetiologische untersuchungen über der fieberhaften, herpes serologische untersuchungen zur 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and irf- knockout mice irf- /icsbp and irf- cooperatively stimulate mouse il- promoter activity in macrophages in vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus type i interferons enhance production of ifn-gamma by nk cells interferon (ifn)-alpha/beta, interleukin (il)- and il- coordinately induce production of ifn-gamma during infection with herpes simplex virus type differential secretion of tnf-alpha and ifn-gamma by human peripheral bloodderived nk subsets and association with functional maturation the role of stat in species-specific regulation of th cell development by type i ifns interleukin and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin is a physiologic antagonist il- alpha and tnf-alpha are required for il- -induced development of th cells producing high levels of ifn-gamma in balb/c but not c bl/ mice inhibition of replication of herpes simplex virus in mouse macrophages by interferon alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type mathematical analysis demonstrates that interferons-beta and -gamma interact in a multiplicative manner to disrupt herpes simplex virus replication effect of interferon on replication of herpes simplex virus types and in human macrophages mechanism of inhibition of hsv- replication by tumor necrosis factor and interferon gamma synergistic anti-hsv effect of tumor necrosis factor alpha and interferon gamma in human corneal fibroblasts is associated with interferon beta induction synergistic anti-herpes effect of tnf-alpha and ifn-gamma in human corneal epithelial cells compared with that in corneal fibroblasts daubener w: inhibition of human herpes simplex virus type by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine , -dioxygenase interleukin- (il- ) and il- are important in innate defense against genital herpes simplex virus type infection in mice but are not required for the development of acquired gamma interferon-mediated protective immunity interleukin- -and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type il- and il- act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system reduced severity of hsv- -induced corneal scarring in il- -deficient mice il- suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis therapeutic protective effects of il- combined with soluble il- receptor against established infections of herpes simplex virus type in thermally injured mice requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin administration an absolute and restricted requirement for il- in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections interleukin- and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type infection in the absence of t cells, natural killer cells protect from mortality due to hsv- encephalitis protective immunity to genital herpes simpex virus type infection is mediated by t-bet the role of natural killer cells in protection of mice against death and corneal scarring following ocular hsv- infection kinetics of cytokine production in the cornea and trigeminal ganglion of c bl/ mice after corneal hsv- infection human natural killer cell deficiencies and susceptibility to infection nitric oxide as a secretory product of mammalian cells herpes simplex virus type synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha production of key molecules by ocular neutrophils early after herpetic infection of the cornea requirement for type no synthase for il- signaling in innate immunity absence of tumour necrosis factor facilitates primary and recurrent herpes simplex virus- infections macrophage control of herpes simplex virus type replication in the peripheral nervous system nitric oxide and hsv vaginal infection in balb/c mice interferon (ifn)-gamma and herpes simplex virus/tumor necrosis factor-alpha synergistically induce nitric oxide synthase in macrophages through cooperative action of nuclear factor-kappa b and ifn regulatory factor- requirement for transcription factor irf- in no synthase induction in macrophages endothelial interferon regulatory factor cooperates with nf-kappa b as a transcriptional activator of vascular cell adhesion molecule nf kappa b and interferon regulatory factor physically interact and synergistically induce major histocompatibility class i gene expression interferon regulatory factor- physically interacts with nf-kappa b in vitro and inhibits nf-kappa b induction of major histocompatibility class i and beta -microglobulin gene expression in transfected human neuroblastoma cells complex formation of the interferon (ifn) consensus sequence-binding protein with irf- is essential for murine macrophage ifn-gamma-induced inos gene expression does nitric oxide play a critical role in viral infections? inhibition of viral replication by interferon-gammainduced nitric oxide synthase interferon-gamma induced type i nitric oxide synthase activity inhibits viral replication in neurons inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites nitric oxide and macrophage antiviral extrinsic activity evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type replication early inhibition of nitric oxide production increases hsv- intranasal infection role of nitric oxide synthase type in acute infection with murine cytomegalovirus mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced th cell responses phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase epr characterization of molecular targets for no in mammalian cells and organelles dna strand breakage, activation of poly (adp-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. formation of novel nitrogen-containing oxidized lipid derivatives altered immune responses in mice lacking inducible nitric oxide synthase rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase -deficient mice osterhaus ad: inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus interleukin- -mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta null mouse effect of the deletion of us and us from herpes simplex virus type on immune responses in the murine vagina following intravaginal infection small amounts of exogenous il- increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type herpes simplex virus type infection of macrophages impairs il- -mediated inhibition of no production through tnfalpha-induced activation of nf-kappab il- -induced stat suppresses ifngamma-stimulated stat -dependent transcription in mouse macrophages impaired il- -mediated functions of macrophages in stat -deficient mice structure and regulation of the human interferon regulatory factor (irf- ) and irf- genes: implications for a gene network in the interferon system structurally similar but functionally distinct factors, irf- and irf- , bind to the same regulatory elements of ifn and ifn-inducible genes absence of the type i ifn system in ec cells: transcriptional activator (irf- ) and repressor (irf- ) genes are developmentally regulated recognition dna sequences of interferon regulatory factor (irf- ) and irf- , regulators of cell growth and the interferon system b lymphocyte-induced maturation protein (blimp)- , ifn regulatory factor (irf)- , and irf- can bind to the same regulatory sites inhibition of no production in macrophages by il- is counteracted by herpes simplex virus infection through tumor necrosis factor-alpha-induced activation of nk-kappa b interleukin- and interleukin- modulate nuclear factor kappab activity and nitric oxide synthase- expression in theiler's virus-infected brain astrocytes interaction of interferon regulatory factor- and nuclear factor kap-pab during activation of inducible nitric oxide synthase transcription i wish to thank all members of the group for highly constructive discussions and especially søren c. mogensen for critical review of the manuscript. for excellent technical help with the electron microscopy i thank ruth nielsen. furthermore, i wish to thank the department of clinical microbiology, aarhus university hospital, skejby and department of medical microbiology and immunology, university of aarhus for their hospitality and support. key: cord- - yc ribg authors: morehouse, zachary p.; proctor, caleb m.; ryan, gabriella l.; nash, rodney j. title: a novel two-step, direct-to-pcr method for virus detection off swabs using human coronavirus e date: - - journal: virol j doi: . /s - - -y sha: doc_id: cord_uid: yc ribg background: currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (pcr) based assays. this process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a pcr assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. methods: using human coronavirus e (hcov- e) as a model system, we spiked swabs in vitro for proof-of-concept testing. swabs were spiked in serial dilutions from . × ( ) to . × ( ) copies/ml and then placed in ml tubes with viral transport media (vtm) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. after homogenization, μl of lysate was processed using rt-qpcr for amplification of the nucleocapsid (n) gene, qualifying viral detection. results: hcov- e in vitro spiked swabs were processed in a novel two-step, direct-to-pcr methodology for viral detection. after running swabs, we confidently determined our limit of detection to be . × ( ) viral copies/ml with . % sensitivity. conclusion: we have proven that the shaker-mill homogenization-based two-step, direct-to-pcr procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in pcr for the detection of hcov- e. this finding allows for reductions in the time and resources required for pcr based virus detection in comparison to the traditional extraction-to-pcr methodology. as the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. herein, we are proposing a novel method for viral pathogen detection off swabs as an improvement or alternative to the current pcr based assays commonly used for viral detection [ , ] . the traditional protocol for preparing a sample for pcr based detection often involves procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify its nucleotides, and then amplify the purified genetic material via pcr for detection of a gene product needed to confirm the patient's suspected diagnosis [ ] . in the face of the covid- pandemic, attempts have been made to perform pcr based diagnostics for sars-cov- with reduced time and cost, especially as the plastics and reagents needed for traditional viral nucleotide extractions have become scarce in the face of an exponentially increased global demand [ ] [ ] [ ] [ ] . though some success has been seen with thermal and enzymatic digestion attempts on viral samples to expose the genetic material for extraction-less pcr detection, no method has shown high viral lysis in under min when dealing with clinical concentrations of virus on swabs [ , , ] . shaker-mill homogenization as a form of mechanical lysis has been proven time and again as a successful method for disrupting tissues, microorganisms, and biologic samples for downstream molecular analysis; however, these downstream applications often require the use of additional purification, isolation, or extraction procedures before those analyses can be completed [ ] [ ] [ ] [ ] . while, shaker-mill homogenization procedures have not yet been applied directly to diagnostic technologies, its ability to lyse organisms and tissues far tougher than a virus have been well categorized [ ] [ ] [ ] [ ] . building off the increased need for novel viral diagnostic methodologies, which reduce the resources and time required for accurate viral pathogen detection, we felt it was time to examine the capabilities of shaker-mill homogenization for viral lysis to be used for downstream detection. using human coronavirus e (hcov- e) as our model organism, we developed a novel two-step methodology of optimized shaker-mill homogenization parameters that allowed for direct-to-pcr viral detection. hcov- e is an enveloped, positive-sense, singlestranded rna virus [ ] . as a known human pathogen, it hcov- e is one of the four most common circulating coronaviruses associated with mild to moderate respiratory illness globally [ ] . it is commonly included in commercial respiratory viral panel screening as a source of the common cold [ ] . hcov- e was the chosen model for this project as a biosafety level pathogen with similar genetic and protein composition to the current sars-cov- virus for developing novel diagnostic approaches, until the process is proven in vitro [ , ] . the linear genome and enveloped structure of hcov- e allows for this virus to be a sufficient in vitro substitute for sars-cov- during the early stages of methodology development described within this manuscript [ ] . no. - ), incubated at °c with % co [ ] . the cell culture supernatant was harvested at h post infection when % cytopathic effect (cpe) was observed. viral transport media (vtm) was produced following the us cdc (atlanta, ga, usa) guidelines, found freely available on their website for formulation of vtm in a laboratory as an alternative to commercial vtm purchases. ml of hanks balanced salt solution (hbss) x with calcium and magnesium ions (no phenol red) (fisher science, cat. no. sh ) was supplemented with % heat inactivated fetal bovine serum (gemini bioproducts, cat. no. - ). mg of gentamicin (gemini bioproducts, cat. no. - p) and μg of amphotericin b (gemini bioproducts, cat. no. - p) was added to the mixture and mixed thoroughly to create a final product of viral transpot media, % fbs, μg/ml gentamicin, . μg/ml amphotericin b. this viral transport media was used for storage and processing of all swab samples in this manuscript. sterile cotton swabs (fisher science, cat. no. - - ) were submerged for s in viral solutions ranging from . × to . × viral copies/ml [ ] . the swabs were exposed in a serial dilution pattern, with three swabs being exposed at each concentration log to evaluate the detection capabilities of this method. the saturated swabs were then placed in a ml screw capped tube (omni international, cat. no. - ) prefilled with ml of viral transfer buffer [ ] . the stem of the swab was then broken off at a level even with the top of the tube to allow for the cap to be screwed on for transporting and processing. the samples were prepared at °c and then incubated for h at °c prior to processing. to maintain optimal levels of biosafety, the following shaker-mill processing was completed in a biosafety cabinet to protect the user from any potential aerosol production during processing. twenty-four ml screw cap tubes containing the virally spiked swabs were processed on the omni bead ruptor elite (omni international, cat. no. - e) for s at . m/s. this processing generated froth within the tube which was allowed to settle prior to removal of μl of lysate for rt-qpcr (fig. ) . hcov- e nucleocapsid gene (n gene) was selected as a target for rt-pcr from vabret et al. [ , ] . the n gene was targeted with forward primer ′-aggcgcaa gaattcagaaccagag- ′ and reverse primer ′-agcaggactctgattacgagaaag- ′ [ ] . μl of sample lysate was added to create a final reaction volume of μl using the proportions of primers, sample, sybr, rt, and depc-treated h o as laid out in the new england biologics luna rt-qpcr kit (neb, cat. no. e s). amplification of lysate was performed for cycles and the resulting amplicons were loaded into a % agarose (bio-rad, cat. no. ) gel for product visualization. out of abundance of caution, the loading of the pcr plate with viral lysate should be completed in a biosafety cabinet to protect the user from any potentially viable virus particles remaining following shaker-mill homogenization. hcov- e was quantified with standard plaque assay protocols [ ] . once the cells achieved % confluence, μl of hcov- e stock was added to the media of the first well. the media was gently mixed and μl of the infected media was transferred into the adjacent well. this was repeated to create serial dilutions throughout the plate. after h of incubation, the virally infected media was removed from each well and replaced with ml of dmem infused with % heat inactivated fbs and % agarose (bio-rad, cat. no. ). the plate was incubated for an additional days at °c with % co and plaques were counted to determine viral concentration in plaque forming units/ml (pfu/ml). we have successfully demonstrated that shaker-mill homogenization using the omni bead ruptor elite fig. graphical depiction of general two-step, direct-to-pcr in vitro testing method that was employed in all experiments discussed in this manuscript fig. two-step, direct-to-pcr results from serial dilution of viral concentration spiked onto swabs for limit of detection testing. red lines represent . × viral copies/ml spiked swabs. brown lines represent . × viral copies/ml spiked swabs. pink lines represent . × viral copies/ml spiked swabs. navy lines represent . × viral copies/ml spiked swabs. teal lines represent . × viral copies/ml spiked swabs. olive lines represent . × viral copies/ml spiked swabs. black lines represent . × viral copies/ml spiked swabs. green lines represent virus free, negative control swabs provides sufficient viral lysis from spiked swabs to allow for viral detection via direct rt-qpcr of the lysate. using this novel method (fig. ) at our optimized run parameters of . m/s for s, we found the lower limit of reliable detection to be . × viral copies/ml (figs. , ). this limit of detection was evaluated for reproducibility by testing in vitro spiked swabs from two different viral stock solutions, using rt-qpcr (fig. ) . the results were confirmed via amplicon visualization (fig. ) and demonstrated a sensitivity of . % when processing with this two-step, direct-to-pcr method. in addition to the strong method sensitivity shown with in vitro testing, we also consistently showed greater than % viral lysis after shaker-mill homogenization for s at . m/s. plaque assays were used to determine percent lysis following homogenization as shown with decreased pfu counts between stock and lysate ( table ). the cost, time, and availability of current pcr assays have been considered prohibitive measures in the timely diagnosis and subsequent treatment of viral infections [ ] . there is a critical need for the development of diagnostic technologies that help to improve access and reduce cost-this is especially important for increasing the treatment of infectious diseases in socioeconomically disenfranchised populations. while still in its infancy, it is our hope that this novel methodology can be applied as one potential solution for this need. the authors acknowledge that there are crucial next steps for clinical validation of this methodology but feel that the presented data is compelling and provides a strong starting point for this to occur. while these studies was completed using hcov- e as an in vitro model for methodology development geared towards sars-cov- applications, it is also our hope that this methodology can be further evaluated at both the benchtop and the bedside to be applied to other respiratory viruses also commonly tested for via nasopharyngeal or oropharyngeal swabs and pcr based detection. hcov- e n transcript detection using rt-qpcr following shaker-mill homogenization off swabs spiked in vitro with . × viral copies/ ml. orange lines represent viral stock spiked swabs at . × viral copies/ml. grey lines represent viral stock spiked swabs at . × viral copies/ml. the green line represents a negative control, virus-free swab in addition to the improvements to access of care, this two-step, direct-to-pcr methodology also has the potential to improve laboratory safety. as shown in table , shaker-mill homogenization results in greater than % lysis of the virus in a sample. this high percentage of viral lysis demonstrates that there would be few, if any, viable viral particles left in the sample tube when the laboratory technician opens it for transfer to the rt-qpcr reaction. by using our two-step, direct-to-pcr methodology, the lab technician is also able to significantly reduce exposure to viral particles that would normally be increased when opening and closing sample tubes and adding buffers during traditional nucleic acid extraction processes. the low levels of viable and infectious viral particles when the sample is opened result in decreased exposure potential through laboratory accidents or aerosolization during sample opening that could result in laboratory acquired infections. in contrast to the many benefits provided by this novel methodology, the authors do acknowledge that introducing a new approach to existing methods is difficult. we expect to see resistance from laboratories with currently validated testing methods, researchers with years of experience using the traditional extraction-to-pcr process, and other commercial entities involved in the currently accepted processes for pcr based viral diagnostics. however, it is our opinion that when advancements in technology and methodology can be made that will improve access to care, reduce cost, and improve safety, it is our responsibility to the public to explore these options. in the face of increasing virally caused infections globally, it is our duty to do everything we can to shake every tree and turn over every stone to prepare society to combat these diseases. we have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in pcr based assays for the detection of virus. this novel two-step, directto-pcr method for viral detection off swabs has shown a lower limit of reliable detection at . × viral copies/ml with . % sensitivity in vitro when screening for hcov- e. herein, we have demonstrated the success of this methodology in vitro and propose it as a novel approach to viral detection that allows for decrease run time in comparison to traditional pcr based viral detection assay protocols, as well as a reduction in the materials needed for successful viral detection. bypassing standard extraction methods, while maintaining a lower limit of detection one log below the reported viral load on clinically obtained swabs positive for coronavirus [ ] , we demonstrated that this novel method has warranted further clinical evaluation for its potential to reduce the cost and time needed for each test. direct diagnosis of human respiratory coronaviruses e and oc by the polymerase chain reaction detection of the human coronavirus e, hku , nl , and oc between and in yamagata rt-qpcr testing of sars-cov- : a primer perforrmance of abbott id now covid- rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution a novel reverse transcription loopmediated isothermal amplification method for rapid detection of sars-cov- an alternative workflow for molecular detection fo sars-cov- -escape from the na extraction kit-shortage rapid implementation and validation of a cold-chain free sars-cov- diagnostic testing workflow to support surge capacity fast sars-cov- detection byrt-qpcr in preheated nasopharyngeal swab samples assessing species biomass contributions in microbial communities via metaproteomics a conserved rna seed-pairing domain directs small rna-mediated stress resistance in enterobacteria mapping interactions of microbial metabolites with human g-protein-coupled receptors treatment of human immunodeficiency virus infection with tenofovir disoproxil fumaratecontaining antiretrovirals maintains low bone formation rate, but increases osteoid volume on bone histomorphometry host and sources of endemic human coronaviruses properties of coronavirus and sars-cov- characterization of a human coronavirus (strain e) c-like proteinase activity viral load of sars-cov- in clinical samples nasopharyngeal swab collection in the suspicion of covid- clinical validation of a sars-cov- real-time reverse transcription pcr assay targeting the nucleocapsid gene two detailed plaque assay protocols for the quantification of infectious sars-cov- overcoming barriers in hpv vaccination and screening programs publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge pete tortorelli, karl jahn, and erik masefield for their financial support and organizational support of the experiments needed for this publication. additionally, we would like to acknowledge rachel true, rachel nash, and leah proctor for their continued support of our work.authors' contributions zp morehouse: conceptualization, methodology, validation, formal analysis, investigation, data curation, writingoriginal draft, review, and editing. cm proctor: methodology, validation, formal analysis, investigation, data curation, writingreview and editing. gl ryan: data curation, writingreview and editing. rj nash: conceptualization, methodology, investigation, data curation, writingreview and editing, supervision, funding acquisition. the author(s) read and approved the final manuscript. this work was supported solely through private funding by omni international inc.availability of data and materials all data supporting this manuscript can be accessed by reaching out to the corresponding authors rj nash and zp morehouse directly at rnash @gsu. edu and moreho @msu.edu.ethics approval and consent to participate because no animals or patients were involved in this study, ethics approval is not applicable in this section. not applicable.competing interests zp morehouse, cm proctor, gl ryan, and rj nash all are currently employed by omni international inc. in some capacity but have no personal financial interests in the success of the company. key: cord- - gd w kn authors: beer, christiane; pedersen, lene title: amphotropic murine leukemia virus is preferentially attached to cholesterol-rich microdomains after binding to mouse fibroblasts date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: gd w kn background: we have recently shown that amphotropic murine leukemia virus (a-mlv) can enter the mouse fibroblast cell line nih t via caveola-dependent endocytosis. but due to the size and omega-like shape of caveolae it is possible that a-mlv initially binds cells outside of caveolae. rafts have been suggested to be pre-caveolae and we here investigate whether a-mlv initially binds to its receptor pit , a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains. results: here, we show that a high amount of cell-bound a-mlv was attached to large rafts of nih t at the time of investigation. these large rafts were not enriched in caveolin- , a major structural component of caveolae. in addition, they are rather of natural occurrence in nih t cells than a result of patching of smaller rafts by a-mlv. thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (vsv-g) pseudotyped mlv particles showed the same pattern of large rafts as cells incubated with a-mlv, but vsv-g pseudotyped mlv particles did not show any preference to attach to these large microdomains. conclusion: the high concentration of a-mlv particles bound to large rafts of nih t cells suggests a role of these microdomains in early a-mlv binding events. retroviral vectors carrying the envelope protein of amphotropic murine leukemia virus (a-mlv) are some of the most widely used retroviral vector pseudotypes in gene therapy trials. achievement of controlled but efficient gene delivery will, however, depend on a detailed insight into virus biology. we have previously shown that a-mlv entry is closely associated with cholesterol-rich microdomains like rafts and caveolae [ ] and that a-mlv envelope protein is associated with rafts in infected cells suggesting a possible role of rafts in a-mlv assembly [ ] . it has also been shown for other viruses that rafts and/or caveolae are important for their entry and assembly [ ] [ ] [ ] [ ] [ ] [ ] ; specifically, has caveola-mediated entry been shown for, e.g., sv [ ] , echovirus [ ] , and human coronavirus e [ ] . both domains consist of high concentrations of cholesterol, sphingomyelin, ganglioside gm , and other saturated lipids [ , ] but in contrast to rafts do caveolae build omega-shaped invaginations within the plasma membrane of cells [ ] . the unique lipid composition of rafts and caveolae leads to the specific incorporation or exclusion of proteins in these domains thereby creating distinct microenvironments for cellular processes [ , ] . studying sv entry it was found that viral entry via caveolae occurs through an endocytic mechanism and that it -in comparison to an endocytic entry via clathrin-coated pits -is a cholesterol-dependent, ph-independent, and slow process [ ] . we also found these hallmarks of caveolae-mediated entry when studying a-mlv entry of fibroblastic cells [ ] . association of the viral receptor with caveolae would seem essential for viral entry through caveolae and our previous investigations also showed that the a-mlv receptor protein pit , a sodium-dependent phosphate transporter, is able to directly associate with caveolin- (cav- ) [ ] , one of the major structural proteins of caveolae [ ] . however, the omega-like shape of caveolae and their average size of around nm would suggest that a-mlv with its diameter of about nm binds outside of caveolae. as rafts are suggested to be pre-caveolae [ ] and a large fraction of the a-mlv receptor pit was found associated with cholesterol-rich microdomains [ ] , we have here investigated if rafts and caveolae are involved in the early steps of a-mlv binding. first, we wanted to investigate if a-mlv binds to cholesterol-rich microdomains. therefore, nih t cells were incubated for hours at °c with fluorescently labeled a-mlv (gagyfp a-mlv) containing a nucleocapsid protein fused with yellow fluorescence protein (yfp) [ ] . after subsequent washing and fixation, the cells were incubated with fluorescently labeled cholera toxin (ctx). this is a standard procedure for staining of cholesterolrich microdomains since ctx binds specifically to gm , a marker of rafts and caveolae [ ] . as shown in figure a , cell-bound a-mlv showed a pronounced attachment to large gm -positive microdomains. as gm is a general marker for cholesterol-rich microdomains, we investigated if these regions of preferred a-mlv binding were also enriched in caveolin- (cav- ), a major structural protein of caveolae. nih t cells were incubated with gagyfp a-mlv particles, washed, fixed, and permeabilized. subsequently, the cells were stained for cav- and investigated using confocal microscopy. as expected a part of gagyfp a-mlv particles co-localized with cav- could be observed, however, cav- was not enriched at the favored binding sites of gagyfp a-mlv (fig. b) . the same was true for gagyfp a-mlv bound to nih t cells stably expressing a cav- mred fusion protein (fig. c) . from these data, we suggest that rafts rather than caveolae are involved in the early steps of a-mlv binding. interestingly, in many investigated cells the stained cholesterol-rich microdomains appeared as large patched regions within the plasma membrane of the cells (fig. a) . as the cells were fixed before staining with ctx, we could exclude a patching of smaller rafts due to ctx binding. however, it is known, that binding of ligands or viruses to their raft-associated receptor can lead to patching of smaller raft domains [ , ] . we therefore investi-gated whether virus binding lead to patching of gm -rich microdomains. as a control, we included vsv-g pseudotyped gagyfp mlv particles (here referred to as gagyfp vsv); vsv enters cells via clathrin-coated pits [ ] and binding of these viral particles to the cells should therefore not lead to patching of cholesterol-rich microdomains. thus, vsv or a-mlv pseudotypes of gagyfp mlv cores were added to nih t at °c, and after minutes the cells were washed, fixed, and stained for gm with fluorescently labeled ctx. confocal microscopy revealed that large cholesterol-rich microdomains were present in cells incubated with both vsv and a-mlv (fig. ) . the same was true for nih t cells incubated with viral like particles lacking viral envelope proteins (data not shown). but in comparison to a-mlv, neither vsv nor viral like particles lacking viral envelope proteins showed preferential attachment to large rafts. thus, while binding of a-mlv to the large raft regions seems to be a-mlv envelope specific, it did not lead to the formation of the large rafts. as rafts are enriched in cholesterol and cholesterol has been shown to be important for a-mlv entry [ ] , we wanted to investigate, if cholesterol was important for the preferential binding of a-mlv to the large raft regions. therefore, we treated nih t cells with mm methylbeta-cyclodextrin (mbcd), which is known to extract cholesterol out of the plasma membrane of eukaryotic cells [ ] . after this treatment, the cells were incubated with gagyfp a-mlv for min at °c, washed, fixed, and stained for gm with fluorescently labeled ctx. although we have previously shown that this treatment is sufficient to extract up to percent of the plasma membrane cholesterol of nih t cells [ ] , large raft regions were still present (fig. ). in addition, a-mlv showed the same binding pattern as in the experiments in figures and demonstrating that depletion of cholesterol alone was not sufficient to prevent a-mlv binding to the large raft regions. the a-mlv replication cycle is closely associated with cholesterol-rich microdomains like rafts and caveolae. we have previously shown that a-mlv can enter mouse fibroblasts through caveola-mediated endocytosis and that the a-mlv envelope protein is associated with rafts in infected cells [ , ] . here, we have furthermore demonstrated that rafts are involved in a-mlv binding. using confocal microscopy we found that a-mlv binds preferentially to large gm -positive membrane regions of nih t cells, which are most likely rafts. cav- staining of nih t cells as well as nih t cells stably expressing a cav- mred fusion protein revealed that cav- was not enriched at the favored binding sites of a-mlv. interest- a-mlv binds preferentially to large rafts figure a-mlv binds preferentially to large rafts. a) nih t cells were incubated with gagyfp a-mlv (green) for hours, fixed, and gm was stained with fluorescently labeled ctx (red). b) nih t cells were incubated with gagyfp a-mlv (green) for hours and fixed. the cells were permeabilized with triton x- and cav- was stained (red). c) nih t cells stably expressing cav- mred fusion protein (red) were incubated with gagyfp a-mlv (green) for hours and fixed. clusters of viral particles as those found bound to large rafts in a are labelled with arrows. all pictures were taken using confocal microscopy. ingly, the large rafts were not a result of virus induced aggregation of smaller rafts (raft patching). nih t cells incubated together with vsv or with viral particles lacking a viral envelope protein had the same large gm -positive domains as cells incubated with a-mlv. as vsv enters and infects cells via clathrin-coated pits [ ] , binding of vsv should not lead to any patching of rafts. therefore, we conclude that the presence of large rafts in nih t cells is not a result of a-mlv binding. raft patching is especially known from investigations of the t cell receptor (tcr) and the t cell coreceptor cd . it has been shown that crosslinking of tcr and cd by antibodies as well as with gagyfp a-mlv (green) for min, fixed, and stained for gm with fluorescently labeled ctx (red). clusters of viral particles bound to large rafts are labelled with arrows in b). c), and d) nih t were incubated with vsv (green) for min, fixed, and stained for gm with fluorescently labeled ctx (red). all images were taken using confocal microscopy. a) and c) are merged images, b) and d) show only gagyfp a-mlv or gagyfp vsv particles from a) and c), respectively. incubation of t cells with ctx lead to raft patching [ ] [ ] [ ] . as we fixed the cells prior to staining with ctx, we can exclude that the large rafts are artifacts caused by the staining procedure. it should also be noted that large rafts were not present in other cell lines like t (human kidney cell line) or mdtf (mus dunni tail fibroblasts) regardless of a-mlv binding (data not shown). the origin of the large rafts in nih t cells is not known. however, raft patching occurs mainly through ligandreceptor interactions or other protein-protein interactions [ , ] and it is possible that proteins from serum or the presence of a prominent extracellular matrix associated with rafts could lead to raft patching and the appearance of large rafts in nih t cells. raft patching in nih t cells induced by pronounced protein-protein interactions would indeed explain the resistance of these large domains to extraction of plasma membrane cholesterol with mbcd. thus, it is likely that a complex and rigid pro-tein network could preserve large rafts from disintegration even in the absence of cholesterol. in addition, a-mlv binding to large rafts was independent of plasma membrane cholesterol indicating that the a-mlv receptor pit or other virus interacting proteins were still present in these regions. all vector particles used in the present study were produced by transient transfection of t cells and since a-mlv but not vsv or viral particles lacking viral envelope proteins showed favored binding to large rafts, we conclude that this is due to the presence of a-mlv envelope protein on the particles and therefore most likely due to the presence of pit in these regions. furthermore, in comparison to a-mlv only a small amount of vsv particles were cell-bound at the time of investigation. this is probably due to the faster entry of vsv into cells since we have previously shown that vsv particles enter nih t cells times faster than a-mlv [ ] . in addition, to ensure that the reason for the observed differences in a-mlv and vsv binding was not due to a lesser amount of a-mlv binding to large rafts is independent of extraction of plasma membrane cholesterol figure a-mlv binding to large rafts is independent of extraction of plasma membrane cholesterol. nih t cells were treated with mm mbcd, washed, and incubated with gagyfp a-mlv (green) for min. subsequently, the cells were washed, fixed, and stained for gm using fluorescently labeled ctx (red). images were taken using confocal microscopy. vsv particles we also have incubated cells with lesser quantities of a-mlv. as expected, the amount of cellbound a-mlv decreased but a high amount of the cellbound particles were still found attached to large rafts (data not shown). while we previously demonstrated that the major entry route of a-mlv in nih t cells is via caveola [ ] , we here found that the vast majority of cell-bound a-mlv virions associated with gm -positive regions and not with cav- positive regions. indeed, the amount of a-mlv particles associated with large rafts within minutes of virus exposure suggests that rafts are involved in early events of a-mlv binding and that a-mlv virions bind to the cells outside of caveolae and subsequently associate with caveolae in the entry process. furthermore, in agreement with the slow infection kinetic of a-mlv these data also suggest that transport of a-mlv from rafts to caveolae is a limiting step in a-mlv entry. taken together, our results show that a-mlv binds preferentially to large rafts in nih t suggesting involvement of these microdomains in early steps of a-mlv binding. nih t cells (atcc crl- ) were propagated in dulbecco's modified eagle's medium (dmem) supplemented with glutamine and % newborn calf serum (ncs). t cells (atcc crl- ) were propagated in dmem supplemented with glutamine and % fetal calf serum (fcs). all cells were grown at °c, % co and % humidity. to stably express a cav- mred fusion protein nih t cells were co-transfected with a construct encoding the cav- mred fusion protein [ ] and the psv pac plasmid encoding puromycin resistance gene [ ] . after days of selection with medium containing µg/ml puromycin, the cells were propagated in normal cultivation medium and used for investigations. for production of gag-yfp a-mlv or gagyfp vsv particles, t cells were seeded in t flasks and grown to % confluence. the cells were transiently co-transfected with a gag-yfp construct, containing moloney mlv gaggene encoding viral structural proteins and an yfp-tagged nucleocapsid protein [ ] , phit (β-galactosidase encoding retoviral vector) [ ] , and a phit derived vector encoding the a-mlv ( a isolate) envelope or a plasmid encoding the vsv glycoprotein (clontech). fortyeight hours after transfection, the supernatants were filtrated ( . µm) and stored until use at - °c. to extract cholesterol out of the plasma membrane, nih t cells were overlaid with mm mbcd (sigma). after min at °c, the cells were washed once with cell culture medium and used in virus binding studies. nih t cells were seeded on chamber slides (nunc) and grown to % confluence. the cells were incubated with gagyfp a-mlv or gagyfp vsv for . hour or hours as indicated. in some experiments, the cells were treated with mbcd before incubation with a-mlv. after binding of the viruses, the cells were washed with pbs, immediately overlaid with % paraformaldehyde, and incubated for min at rt. the fixed cells were washed with pbs, blocked with pbs containing % horse serum and % bovine serum albumin, and incubated with an antibody against cav- (bd bioscience and transduction laboratories). subsequently, the cells were overlaid with alexa fluor labelled secondary antibody (molecular probes), washed in pbs, and the slides were mounted with immunofluorescence mounting medium (dako). for staining of gm , the cells were blocked with pbs containing % horse serum and % bovine serum albumin and incubated with alexa fluor -conjugated cholera toxin ( µg/ml) (molecular probes). the confocal images were captured with a leica tcs sp confocal microscope (leitz). yfp and rhodamine were excited individually using argon laser nm line and green helium neon laser nm line, respectively. the two single-color images were subsequently merged into an rgb-image. brightness and contrast were adjusted. caveola-dependent endocytic entry of amphotropic murine leukemia virus amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains bound simian virus translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er evidence for budding of human immunodeficiency virus type selectively from glycolipid-enriched membrane lipid rafts measles virus assembly within membrane rafts internalization of echovirus in caveolae human coronavirus e binds to cd in rafts and enters the cell through caveolae functional rafts in cell membranes structure and function of membrane rafts the caveolae membrane system murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of gag-gag interactions and membrane association gm -containing lipid rafts are depleted within clathrin-coated pits membrane raft microdomains mediate lateral assemblies required for hiv- infection lipid domain structure of the plasma membrane revealed by patching of membrane components pastan i: alpha -macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane t cell receptor can be recruited to a subset of plasma membrane rafts, independently of cell signaling and attendantly to raft clustering aggregation of lipid rafts accompanies signaling via the t cell antigen receptor lipid raft distribution of cd depends on its palmitoylation and association with lck, and evidence for cd -induced lipid raft aggregation as an additional mechanism to enhance cd signaling selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol expression in mammalian cells of a gene from streptomyces alboniger conferring puromycin resistance a transient three-plasmid expression system for the production of high titer retroviral vectors we want to thank dr. richard pagano for the kind gift of the plasmid encoding the cav- mred fusion protein, dr. mary collins for her kind gift of the gagyfp plasmid, and dr. aleksandra rojek for her help with the confocal microscope. the work presented in this article was funded by the german academy of natural scientists leopoldina (bmbf-lpd / - ) (c.b.), the lundbeck foundation, the novo nordisk foundation, the danish medical research council (grant - - ), and the carlsberg foundation. the author(s) declare that they have no competing interests. both authors conceived of the study and drafted the manuscript. cb carried out the experimental work. key: cord- -xsg j t authors: sainz, bruno; barretto, naina; yu, xuemei; corcoran, peter; uprichard, susan l title: permissiveness of human hepatoma cell lines for hcv infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: xsg j t background: although primary and established human hepatoma cell lines have been evaluated for hepatitis c virus (hcv) infection in vitro, thus far only huh cells have been found to be highly permissive for infectious hcv. since our understanding of the hcv lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support hcv infection. here we show infection of the human hepatoma cell lines plc/prf/ and hep b with cell culture-derived hcv (hcvcc), albeit to lower levels than that achieved in huh cells. to better understand the reduced permissiveness of plc and hep b cells for hcvcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). results: we found that while the early events in hcv infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in plc and hep b cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in plc and hep b cultures compared to huh cell cultures. interestingly, we also observed that interferon stimulated gene (i.e. isg ) expression was significantly and differentially up-regulated in plc and hep b cells following viral infection. conclusions: we conclude that the restrictions observed later during hcv infection in these cell lines could in part be attributed to hcv-induced innate signaling. nevertheless, the identification of two new cell lines capable of supporting authentic hcvcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of hcv in vitro and should aid in further elucidating hcv biology and the cellular determinants that modulate hcv infection. worldwide, between and million individuals are chronically infected with hepatitis c virus (hcv), a positive-strand rna virus that infects the liver [ , ] . although acute infection is typically asymptomatic, % of patients fail to clear the virus resulting in a chronic infection associated with the development of significant liver disease, such as fibrosis, cirrhosis, steatosis, insulin resistance and hepatocellular carcinoma (hcc) [ ] . in fact, hcv-related hcc accounts for over % of hcc cases and over % of liver transplants in the united states. despite this obvious public health burden, there is no vaccine to prevent infection and current interferon-based treatment options have toxic side effects and limited efficacy. the main obstacle that has impeded hcv research and antiviral drug development since its discovery in [ ] has been the lack of a robust infectious cell culture system capable of recapitulating all aspects of the viral lifecycle. although early advancements in the study of hcv were made using surrogate systems [ ] , replicons [ ] [ ] [ ] [ ] and hcv pseudotyped particles (hcvpp) [ ] , it was not until the development of the cell-culture derived hcv (hcvcc) system in that robust hcv infection was finally achieved in vitro [ ] [ ] [ ] . this system was based on the identification of an hcv genotype a molecular clone [ ] , shown to be capable of replicating and assembling infectious particles in cell culture, and the discovery that the human hepatoma huh cell line is permissive for hcv infection. although numerous other human hepatoma cell lines exist, only hepg cells and a few other hepatoma cell lines have been rigorously tested for hcvcc permissiveness to date with varying degrees of success [ , [ ] [ ] [ ] [ ] [ ] [ ] . identification of other cell lines able to support hcv infection would not only expand our current repertoire of cell lines available for the study of hcv, but could also prove useful for the identification of cellular determinants of hcv infection. to identify other cell lines suitable for hcv infection studies, we assembled a panel of hepatic and non-hepatic cell lines and assessed their permissiveness for hcv infection. here we show hcvcc infection (i.e. replication, protein translation and de novo virion production) in human hepatoma cell lines plc/prf/ and hep b. like huh cells, plc cells, a human hepatoma cell line first isolated in the early s [ ] and hep b cells, a human hepatoma cell line derived from a hepatocellular carcinoma isolated from an year old male [ ] , have been previously utilized for hcv entry (hcvpp) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] studies; however, to our knowledge, hcvcc infection and rna replication in these two cell lines in the absence of complementation has not been previously reported. although numerous groups have assessed the permissiveness of multiple cell lines for hcvpp infection [ , ] , since the development of the hcv infectious cell culture system [ ] [ ] [ ] a comprehensive analysis of the permissiveness of human hepatic and non-hepatic cell lines for hcvcc infection has yet to be reported, and the identification of alternate cell lines that support robust hcvcc infection is still warranted. thus, we compiled a panel of human cell lines of hepatic and non-hepatic origin ( table ) that have been routinely used for hcvpp entry [ , , , ] and assessed their permissiveness for hcvcc infection. first, for control hcvpp infections, cells were infected with pseudotyped lentiviruses encoding a luciferase reporter and bearing the glycoproteins e and e of hcv genotype a (jfhpp), hcv e and e of genotype a (h pp) or the vsv g glycoprotein (vsvgpp). at h post infection (p.i.) pseudoparticle entry was assessed by luciferase activity as previously described [ ] . all cell lines were equally permissive for vsvgpp infection ( × - × rlu, data not shown) however, only huh , hep b, plc and hepg cells stably expressing cd (hepg -cd , additional file : figure s ) were permissive for hcvpp infection ( figure a) . all other cell lines tested exhibited less than % of maximum luciferase activity, indicating that these cells are non-permissive for hcvpp entry, which is consistent with previously published reports [ ] . to assess permissiveness for hcvcc infection, all cells were infected with hcvcc at an moi of . ffu/cell and intracellular hcv rna was determined by rtqpcr analysis h p.i. we included chinese hamster ovary (cho-k ) cells as they lack all known hcv entry receptors and therefore serve as a control for background hcv rna levels (i.e. non-specific cell bound hcv) ( figure b , dashed line = copies/μg rna) [ ] . as expected, huh cells were highly permissive for hcvcc and replicated hcv rna to levels of . × ± . × copies/μg rna by h p.i. caco- cell showed minimal to no hcvcc permissiveness, reaching levels of only . × ± . × copies/μg rna. although caco- cells have previously been reported to support suboptimal hcvcc infection [ ] , the low levels of hcv rna (p value > . ) measured in these infected cultures are likely due to receptor-bound hcv similar to that of the non-permissive parental hepg cell line. addition of cd to the hepg cells conferred hcvcc permissiveness, as previously reported [ ] , resulting in increased hcv rna replication of . × ± . × copies/μg rna (p value < . ). notably, statistically significant hcvcc infection was also detected in plc cells and hep b cells. although plc cells were less permissive for hcvpp ( figure a ), plc cells contained -fold higher hcv rna levels ( . × ± . × copies/ μg rna) h after infection with hcvcc compared to hep b cells ( . × ± . × copies/μg rna) ( figure b) , which were as permissive as huh cells for hcvpp infection ( figure a ). taken together, these data indicate that plc and hep b cells are permissive for both hcvpp and hcvcc infection, although their permissiveness for hcvcc and hcvpp infection is reduced to different degrees compared to huh cells, suggesting that plc and hep b cells may differ in their capacity to efficiently support specific [ ] , hep b cells [ ] and hepg -cd cells are hepatoma-derived cells lines which although differ cytologically from one another share similar hepatocytes-like characteristics with huh cells, such as cuboidal epithelial-like morphology with high nucleus-tocytoplasm ratio, containing mono-and bi-nuclei with multiple nucleoli and cytoplasmic granules (additional file : figure s ). our initial screening ( figure ) indicated that these three cells lines also share permissiveness for both hcvpp and hcvcc infection albeit to differing degrees. since viral binding/entry represents the first step in the hcv lifecycle, we first assessed the expression of the known hcv entry factors in these cells by measuring cd , sr-bi, cldn and ocln mrna levels by rtqpcr analysis (figure a ). when compared to huh , no significant difference in mrna expression for the four known hcv entry factors was observed among the four cell lines. we next analyzed the cell surface expression of cd and sr-bi by flow cytometry and cldn and ocln by indirect immunofluorescence (if) as our commercially-available antibodies to these two tight junctions proteins are not amendable to flow cytometric analysis. as shown in figure b , cell surface expression of cd and sr-bi in plc, hep b and hepg -cd cells, as measured by flow cytometry, was comparable to that observed on huh cells. if analysis of cldn and ocln also confirmed cell surface expression of these receptors on the newly identified hcvcc-permissive plc and hep b cells as well as the previously characterized huh [ ] and hepg -cd cells [ ] . taken together, these data indicate that plc and hep b cells, at a minimum, express and localize all four known hcv entry receptors on their cell surface. despite expression all four of the previously established essential hcv entry factors, the plc and hepg -cd cells showed a reduced permissiveness for hcvpp infection ( figure a ) when compared to huh and hep b cells. while varying luciferase activity after parallel hcvpp inoculation is a phenotype we have seen among huh cell lines from different laboratories (additional file : figure s and [ ] ), to functionally test whether these cells express suboptimal levels of any of the known hcv entry factors, we transiently transfected plc, hep b and hepg -cd cells with vectors expressing the four known human hcv receptors and reassessed their permissiveness for hcvpp and hcvcc infection h post-transfection. as shown, expression of hcv entry factors in trans individually ( figure ) or in combination (data not shown) did not enhance hcvpp ( figure a -c) or i. and hcv rna was quantified by rtqpcr, normalized to gapdh and is displayed as hcv rna copies/μg total cellular rna (means ± sd for triplicate samples). significant increases in hcv rna levels compared to cho cells (one-way anova and tukey's post hoc t test) are denoted as * p value < . . dashed line represents background levels of non-specific bound hcv rna. hcvcc ( figure d -f) infectivity of plc, hep b or hepg -cd cells. hence, the % less luciferase activity generated when plc cells were inoculated with hcvpp compared to huh and hep b cells, is not due to suboptimal expression of any of the known hcv entry factors. plc and hep b cells can support equivalent levels of hcv rna replication as huh cells defects in post entry steps, such as reduced hcv rna replication, can negatively affect hcvcc infectivity even if entry is achieved. since both plc and hep b cells appear to be entry competent to reasonable degrees, their more significantly reduced permissiveness for hcvcc infection compared to huh cells may be due to defects in hcv rna replication. to determine their capacity to support hcv rna replication, plc and hep b cells were transfected with hcv genotype a subgenomic (sg a) replicon in vitro-transcribed rna, and g -resistant colony formation was determined as has been previously described for huh [ ] and hepg cells [ ] . hcv sg a replicons could be readily established in both cell lines; however, the ratio of colony formation to μg of in vitro-transcribed rna was considerably lower in both hep b and plc cells compared to huh cells ( figure a ). since this reduction could be attributed to differences in transfection efficiency, we introduced hcv by an alternative means in which plc and hep b cells were infected with medium from huh cells replicating the full length a (fl a) hcv replicon that contains infectious hcvcc capable of conferring g resistance to newly infected cells if entry is achieved and replication is established [ ] . unlike the results obtained following transfection of plc and hep b cells with sg a rna, the number of g -resistant colonies established in both plc and hep b cells infected with hcvcc g was more comparable to that achieved in huh cells ( figure b ), although a % reduction in hep b colony formation was noted (p = . ). we next expanded some of these g -resistant sg a and fl a plc and hep b clones and quantified hcv rna levels in each clone to determine steady-state hcv rna replication levels achieved in each cell line. rtqpcr analysis revealed relatively similar levels of hcv sg a rna replication in all three cell lines, with no significant difference (p value > . ) in mean hcv rna copies/μg intracellular rna observed between huh , plc and hep b g -resistant sg a clones ( figure c ; . × ± . × vs. . × ± . × and . × ± . × , respectively). likewise, no significant difference (p value > . ) in the levels of intracellular hcv rna in fl a hcvcc g -established clones was observed between huh , plc and hep b g -resistant fl a clones ( figure d ; . × ± . × vs. . × ± . × and . × ± . × hcv rna copies/μg, respectively). importantly, since g -resistant colony formation following hcvcc g infection is dependent on both successful viral entry and efficient hcv rna replication, the fact that similar numbers of g -resistant colonies and equal levels of hcv rna replication were achieved in both plc and hep b, compared to huh cells, would indicate that despite individual differences in the efficiency of hcv entry or hcv replication between these cell lines together the cumulative efficiency of hcvcc entry plus subsequent rna replication is relatively equivalent in plc, hep b, and huh cells, thus allowing equivalent stable infection initiation events to occur (i.e. fl a colony formation). however, to rule out the possibility that hcvcc g infection could have selected for a population of plc or hep b cells with enhanced efficiency for viral entry and/or replication, we cured fl a plc and hep b replicon clones by ifntreatment and re-assessed their permissiveness for hcvcc infection. as shown in additional file : figure s , none of the ifn-cured fl a clones showed enhanced permissiveness for hcvcc infection compared to their parental control. plc, hep b and hepg -cd cells are defective for de novo hcvcc production thus far, our results indicate that cumulatively the early events in hcv infection (i.e. entry plus replication initiation) are equivalent or only marginally reduced in plc and hep b, respectively compared to huh cells. therefore, the . and log reduction in hcvcc permissiveness observed for the plc and hep b cell lines, respectively ( figure a ), is likely attributable to a defect (s) in a post replication step, such as assembly and/or egress of de novo hcvcc. to monitor multiple rounds of hcv infection and spread, which is dependent on hcv assembly and egress, we performed a kinetic analysis of hcv infection over the course of days in huh , plc, hep b and hepg -cd cells after infection with hcvcc at a low moi ( figure ). using this approach, we show that intracellular hcv rna levels ( figure a ) in huh cells infected with hcvcc at an moi of . ffu/ cell reached . × ± . × copies/μg rna by one day p.i. and increased exponentially thereafter , -fold reaching a steady-state level of . × ± . × copies/μg rna by day p.i. hcv-infected plc cells showed parallel but slightly reduced hcv rna expansion kinetics during the first h p.i. compared to hcv-infected huh cells; however, by day p.i., hcv rna levels began to decline reaching undetectable levels by day p.i. in hcv-infected hep b cells, following a - log increase in hcv rna levels during the first h p. i., hcv rna reached and maintained a low steady-state level of~ . × ± . × copies/μg rna. similarly hepg -cd cells clearly lacked a notable robust expansion in hcv rna levels after h p.i. and maintained a low steady-state level ( . × ± . × copies/μg rna) until day p.i. likewise, when entry was bypassed by transfecting cells with infectious full-length hcv jfh- in vitro-transcribed rna, a similar profile of rna expansion and kinetics was observed (additional file : figure s ). analysis of hcv-positive cells by immunocytochemical staining of hcv e protein days p.i ( figure b ), indicated that while the majority of cells in the huh culture had become infected, only a small percentage of the cells in the plc, hep b and hepg -cd cultures were infected likely representing the % of cells that where initially infected by the viral inoculum. since viral spread to a large extent is mediated by de novo secretion of infectious hcv virions, we next quantified the level of infectious hcvcc secreted into the supernatants of each infected cell line over the course of days ( figure c ) using a standard hcv foci formation assay as described [ ] . while infectious hcvcc was detectable in hep b and hepg -cd -infected cultures at reduced levels, infectious hcvcc was undetectable (i.e. below the lower limit of the assay) in the culture supernatant of hcv-infected plc cells even at day when the plc cultures had amplified a higher level of intracellular hcv rna than the hep b and hepg -cd cells ( figure c ). were expanded in the presence of μg/ml g and total rna extracted~ weeks post expansion. hcv rna was quantified by rtqpcr, normalized to gapdh and is displayed as mean hcv rna copies/μg total cellular rna ± sd for each clone. to distinguish whether the lack of infectious hcvcc present in the supernatant of hcv-infected plc cells ( figure c ) is due to a defect in i) intracellular hcv rna encapsidation and virion assembly and/or ii) release of de novo infectious hcvcc, we infected huh and plc cells with hcvcc at an moi of . ffu, collected cells and culture medium from infected cultures over the course of days, and quantified intracellular and extracellular hcvcc infectivity titers and viral rna. consistent with the data shown in the day low moi infection experiment ( figure c ), increasing infectious hcvcc levels were detected in the supernatant of hcvinfected huh cells; however, at no time p.i. did we detect infectious hcvcc in the culture supernatants from hcv-infected plc cells ( figure a ). to determine whether non-infectious hcvcc virions were secreted from the infected plc cells, supernatants were treated with s micrococcal nuclease, rna extracted and hcv genome copies quantified by rtqpcr. as shown in figure b , extracellular nuclease-protected hcv rna (i.e. encapsidated viral rna) was detected in culture supernatant harvested from hcv-infected huh cells, and levels exponentially increased over the course of infection, consistent with the production and excretion of de novo infectious hcvcc. in hcv-infected plc cells, however, significantly lower levels of nuclease-protected hcv rna were detected with a modest exponential increase in total copies observed between days and . notably, if the hcv rna present in the plc cell supernatant did reflect infectious hcvcc, the level of infectivity (~ - ffu/ml) would be below the level of detection in our titration assay ( ffu/ml). thus, we can not conclude from this whether the low level of hcv rna secreted by plc cells represents infectious or non-infectious virions. to assess the presence and infectivity of intracellular hcv virions, cells were washed extensively with × pbs, trypsinized, resuspended in % cdmem, pelleted at , rpm for min and resuspended cell pellets subjected to multiple freeze thaws. cell debris was removed by centrifugation and the presence of infectious hcvcc in cleared lysates was determined by foci titration analysis and the presence of non-infectious hcv by rna analysis following s micrococcal nuclease treatment. as expected, increasing levels of infectious hcvcc and nuclease-protected hcv rna were detected in intracellular lysates from hcv-infected huh cells over the course of infection ( figure c and d, respectively) . in hcv-infected plc cells, low levels of infectious hcvcc were detectable only on days and p.i. (figure c) , and similar to the levels of extracellular hcv rna detected in culture supernatants ( figure b ), low levels of nucleaseprotected hcv rna were detected in intracellular lysates, again with a modest exponential increase in total copies observed between days and . together, these data indicate that the ability of plc cells to efficiently encapsidate hcv rna is markedly reduced compared to huh cells, resulting in low to undetectable levels of secreted infectious de novo hcvcc. viruses often induce a cellular innate immune response, mediated by the induction of ifn-stimulated genes (isg), in the infected host cell [ ] . although the cellular isgs and pathways induced by viral infections are generally similar among mammalian cells (reviewed in [ ] ), different cell lines possess different abilities to two μg of total murine carrier rna was added to all samples for normalization. hcv rna values were determined by rt-qpcr and are displayed as hcv copies/μg total carrier rna (means ± sd for triplicate samples). the dashed line represents the background of the assay ( hcv rna copies/μg carrier rna). (c-d) for intracellular infectivity titer and rna analysis, cells were pelleted, re-suspended in complete % cdmem and lysed via multiple freeze/thaws. (c) intracellular infectivity titers, expressed as ffu/ml, were determined on naïve huh - cells. the dashed line represents the lower limit of detection of the assay ( ffu/ml). (d) intracellular hcv rna copies were determined after s micrococcal nuclease treatment by rt-qpcr and are displayed as hcv copies/μg total carrier rna (means ± sd for triplicate samples). the dashed line represents the background of the assay ( hcv rna copies/μg carrier rna). mount specific antiviral responses to the same virus, which can potentially cause differences in permissiveness to viral infections. this has recently been demonstrated by zhao et al., for murine coronavirus infection of different murine cell lines [ ] . to determine if hcv differentially induces the expression of isgs in huh , plc, hep b and hepg -cd cells following infection, we evaluated isg mrna induction by rtqpcr following infection of cells with hcvcc at an moi of . ffu/cell. as shown in figure , isg mrna levels were not up-regulated in hcv-infected huh cells and remained constant throughout the course of infection, indicating that as previously shown [ ] , hcvcc does not induce an efficient innate interferon response in huh cells. in contrast, induction of isg was observed in the other three human hepatoma cell lines following hcvcc infection. specifically, isg mrna levels were up-regulated -and -fold in hep b and hepg -cd cells, respectively, as early as h p.i. and reached a steady state level by days post infection. the highest and most significant induction of isg , however, was observed in hcv-infected plc cells. notably, we have previously shown that ifn-mediated activation of the innate cellular interferon response inhibits hcv rna replication and subsequent production of de novo hcv virions ( [ ] and data not shown), thus the reduced steady-state hcvcc infection levels observed in hep b and hepg -cd cells and the decline of hcv infection observed in plc cells, despite these cells exhibiting comparable infection initiation permissiveness, may be at least in part a consequence of hcv-induced innate immune signaling in these hepatoma cell lines. in this study we characterized a panel of hepatic and non-hepatic cell lines (table ) for hcvcc permissiveness. all non-hepatic cell lines were either refractory or marginally permissive for hcvpp and hcvcc infection (figure ), as previously shown [ , , , , ] ; however, contrary to commonly held perception, all three of the hepatoma cell lines tested were permissive for hcvcc infection, although to varying degrees relative to huh cells. considering the phenotypic differences observed among the hepatoma cell lines (additional file : figure s ), the perception that huh cells are relatively unique in their ability to support hcv infection [ , ] , and based on previous reports demonstrating varying degrees of hcvcc infection in other hepatoma cell lines, such as hepg , heparg, and immortalized human hepatoma (ihh) cells [ , , [ ] [ ] [ ] , ] , we were surprised that hcvcc infection and replication could be readily detected in both hep b and plc cells (figure ). in fact, hcv rna levels exceeding copies/μg rna were detected in plc cells days post infection with hcvcc. when compared directly to huh cells, however, it was apparent that hcvcc infection in both plc and hep b cells was reduced ( . × ± . × vs. . × ± . × and . × ± . × copies/ μg rna, respectively). in addition, our initial screen also showed notable differences in permissiveness for hcvpp infection between these two cell lines. specifically, hcvpp infectivity was~ % lower in plc cells compared to hcvpp-infected huh and hep b cells. however, although significant (p value = . and . for jfhpp and h pp, respectively), we have seen similar variability in permissiveness for hcvpp infection even among fully hcvcc-permissive huh cell lines from different laboratories (additional file : figure s and [ ] ), indicating that this level of hcv entry should be sufficient to support robust hcvcc infection. consistent with this hypothesis, permissiveness for hcvcc infection initiation was notably higher in plc cells compared to hcv-infected hep b cells ( figure b and b) , despite the lower permissiveness of the plcs for hcvpp entry ( figure a ). taken together, the results from our initial screen (figure ) indicated that plc and hep b cells are indeed permissive for both hcvpp and hcvcc infection; however, differences in permissiveness compared to huh cells and between each cell line do exist, and thus we initiated a systematic analysis of plc and hep b permissiveness for hcvcc infection, performing experiments that focused on the different steps of the viral lifecycle (i.e. entry, replication, egress and spread). i. hcv entry. to determine if the differences in hcvpp and hcvcc infection between each hepatoma cell line could be attributed to entry factors already shown to modulate hcv permissiveness in other hepatoma cell lines [ , , [ ] [ ] [ ] [ ] , we examined the expression of four essential hcv entry factors (i.e. cd , sr-bi, cldn and ocln) by rtqpcr, flow cytometric and immunofluorescence analysis ( figure ). interestingly, the data indicated that when compared to the fully permissive huh cell line, no obvious differences in mrna level or cell surface expression of cd , sr-bi, cldn or ocln was observed in plc, hep b or hepg -cd cells. in addition, we over-expressed each of the four hcv entry factors in each cell line since we and others have shown that expression of hcv entry factors in trans can enhance hcvpp and hcvcc entry in cells where hcv permissiveness is low [ , , ] . consistent with the observation that these cell lines already express the necessary levels of the four hcv entry factors, over-expression of cd , sr-bi, cldn or ocln individually or in combination did not significantly increase hcvpp or hcvcc entry in any of the hepatoma cell lines (figure ) . hence, the current data indicate that this panel of hepatoma cell lines expresses the minimum set of known entry factors necessary for efficient hcv entry. however, we cannot rule out the possibility that the cells tested are deficient in another hcv entry factor (e.g. the transferin receptor protein [ ] or the niemann-pick c -like cholesterol absorption receptor [ ] , which we recently showed to be hcv entry factors), entry cofactors (e.g. epidermal growth factor receptor and ephrin receptor a [ ] ) or a post entry host cellular protein required for efficient viral fusion or nucleocapsid uncoating. ii. hcv rna replication. to examine hcv replication independent of inherent differences in plc and hep b cell transfection efficiency, we took advantage of the g -selectable hcvcc g virus [ ] , which can be used to establish hcv fl a replicon cells via direct infection of cells rather than electroporation. using this approach, , and g -resistant colonies were formed in huh , plc and hep b cultures, respectively, weeks following infection with ffu of hcvcc g ( figure b ). additional analysis of hcv rna replication levels in selected fl a clones revealed that comparable levels of hcv rna replication were achieved in all three cell lines ( figure d ). likewise similar levels of intracellular hcv rna were achieved in sg a replicon cell clones ( figure c ). importantly, since g -resistant colony formation following hcvcc g infection is dependent on both successful viral entry and efficient hcv rna replication, the fact that similar numbers of g -resistant colonies and equal levels of hcv rna replication were achieved in both plc and hep b cells, compared to huh cells, indicate that the cumulative efficiency of viral entry plus subsequent rna replication initiation is relatively equivalent between plc, hep b and huh cells. iii. spread and infectious hcvcc production. by performing a day low moi infection assay, we were able to assess the efficiency of viral spread by assessing intracellular hcv rna expansion relative to de novo infectious hcvcc production and by visualizing viral spread directly by immunostaining for hcv-positive cells. analysis of hcv rna replication following infection of each cell line with hcvcc ( figure a ) or electroporation with full-length infectious hcv jfh- in vitro-transcribed rna (additional file : figure s ) revealed that while intracellular hcv rna initially increased in all the infected/transfected cell cultures at different rates, it was only in huh cultures where hcv rna levels continued to increase exponentially past day or post infection/transfection. notably, exponential increase of viral rna in these cultures after day - p.i. would require spread of the virus from the original infected/ transfected cells. consistent with this, hcv e staining on day post-hcvcc infection revealed that the vast majority of huh cells were hcv-positive while little to no spread was evident in the plc, hep b or hepg -cd cell cultures ( figure b ). thus, while infection could be initiated in all these cell lines, only huh cells appeared to support efficient viral spread. since spread of hcv to naïve cells following infection is dependent on secretion of de novo infectious virions, we measured infectious hcvcc in the supernatant of infected cultures and observed detectable but reduced levels of infectious hcvcc in hep b and hepg -cd culture supernatants and undetectable levels of hcvcc in infected plc cultures ( figure c ). low steady-state levels of intracellular hcv rna and infectious virus production in the hep b and hepg -cd cells could result from a defect/bottleneck in any phase of the viral life cycle (e.g. reduced and/or isg inhibition of entry, replication or secretion); however, since the cumulative efficiency of viral entry plus subsequent initiation of hcv rna replication appears to be relatively equivalent between hep b, hepg -cd and huh cells ( figure b ), it is reasonable to speculate that robust hcv infection in hep b and hepg -cd cells may be limited by a decreased capacity for virus production compared to huh cells. of course, in the case of plc cells, there was a clear and severe defect in hcvcc virus production. not only were no infectious particles detected in the supernatant of infected cultures ( figure b and a ), but analysis of hcvcc present within the cell revealed low to undetectable levels of intracellular infectious virions ( figure c ) and comparably low levels of encapsidated hcv rna in both these extra-and intra-cellular samples ( figure b and d) suggesting virion assembly is restricted at or before genome encapsidation. the reduced ability of plc, hep b and hepg -cd cell cultures to support robust steady-state infection levels or spread of hcvcc is likely multifactorial and could be directly due to or influenced by the lack of sufficient levels of a cellular protein necessary for efficient production of infectious progeny virus or a consequence of the presence of an inhibitory factor. i. isg induction. since induction of innate immune signaling can have negative affects on multiple steps in the hcv lifecycle, we assessed the ability of plc, hep b and hepg -cd cells to mount an innate immune response against hcv infection by measuring isg mrna induction, as a representative isg, following hcvcc infection. importantly, while no induction of isg was observed in huh cells, as previously published [ ] , isg mrna was up-regulated in hcvinfected plc, hep b and hepg cells, with plc cells exhibiting the most significant induction following hcvcc infection (figure ) . although it is generally believed that most cultured hepatoma cells generally are impaired for poly(i-c)-and virus-activated ifn responses [ ] , these results are consistent with previous findings that ifn-β promoter activity is more potently induced in hepg and hep b cells than in huh cells following either liposome-mediated transfection of poly (i-c) or sendai virus infection [ ] . relevant to this, a more recent publication by marukian et al. [ ] shows a correlation between innate antiviral gene induction and impaired hcv replication and spread in primary liver cells. likewise, hcv spread was not observed in highly differentiated microscale primary human hepatocyte cultures described by ploss et al., ( ) , who speculated that while the expression of viral proteins in the initially infected cells might be sufficient to blunt intracellular antiviral signaling and allow sustained viral replication, interferon produced by the infected cell may be sufficient to signal to/activate adjacent naïve cells rendering them resistant to infection and thus limiting subsequent viral spread [ ] . thus, the differences in isg induction observed between these cells lines (figure ) may keep hcv replication lower on a per cell basis (hep b and hepg -cd ) and/or at higher levels successfully inhibit viral replication in the infected cells sufficiently to contribute to clearance of the infection (plc). ii. mir- . while isgs are well known negative determinants of hcv permissiveness, numerous positive determinants required for efficient hcv replication have also been identified (reviewed in [ ] ). although overlap between independent studies is not often observed, leaving the relevance of many of these factors to be confirmed, new data from multiple laboratories has recently demonstrated that human micro rna (mir ) is a critical liver-specific factor required for robust hcv infection and secretion [ ] [ ] [ ] [ ] [ ] [ ] . because ifn treatment has been shown to reduce mir expression in huh cells [ ] , it is possible that the enhanced isg induction observed in the other three cell lines may more potently reduce this important positive regulator of hcv infection. likewise, mir has been shown to play an important role in regulating cholesterol homeostasis, fatty acid metabolism and lipogenesis [ ] [ ] [ ] ] , three lipid metabolic pathways shown to be necessary for hcv replication, assembly and secretion. in fact, just prior to submitting this manuscript, evans and colleagues and matsuura and colleagues separately showed that exogenously expressing human mir in hepg or hep b cells, respectively, results in robust hcv replication and infectious virus secretion following hcvcc inoculation or hcv rna transfection [ , ] . hence, lack of adequate mir may also contribute to the reduced hcv permissiveness of certain cell lines. in summary, by testing a panel of human hepatoma and non-hepatoma cells for hcvcc permissiveness we identified new cell lines capable of supporting authentic hcvcc infection (e.g. entry, replication, protein expression and at least for hep b cells, de novo virion production). the permissiveness of plc and hep b cells for hcvcc infection is clearly less robust than that achieved in the highly hcvcc-permissive huh cell lines [ ] and while further studies are needed to definitively and quantitatively assess the host cell factors that limit hcv infection in these cells, these two hepatoma cell lines nonetheless provide new in vitro tools for elucidating the cellular factors that control or mediate hcv infection, particularly those associated with hcv isg induction or those involved in de novo hcvcc production and secretion. in addition, hcv sub-genomic and fulllength replicons have not been previously established in hep b and plc cells [ ] . thus, the clones generated in this study add to the current repertoire of sub-genomic and full-length hcv replicons already available to study specific aspects of the hcv life cycle such as hcv polyprotein processing, replication complex formation and hcv rna replication. expanding our arsenal of reagents and cell lines amendable for the study of hcv should certainly advance our overall understanding of the hcv viral life cycle and the cellular determinants involved, facilitating the potential development of new and more specific hcv antivirals. huh - cells have been previously described [ ] . jfh- cell culture-propagated hcv (hcvcc) viral stocks were obtained by infection of naïve huh - cells at a multiplicity of infection (moi) of . focus forming units (ffu)/cell, using medium from huh - cells electroporated with in vitro-transcribed full length infectious hcv jfh- rna as previously described [ ] . the genotype a infectious jfh- (pjfh- ), the full length (fl) jfh- replicon (pfljfh) and the subgenomic (sg) jfh- replicon (psgjfh) plasmids were kindly provided by dr. wakita (national institute of infectious diseases, tokyo, japan) and have been previously described [ , , ] . the human cd (pee -hucd ), human sr-bi (pzeo_hsr-bi), human cldn (pzeo_cldn ) and human ocln (pcdna . _ocln) expression plasmids have been previously described [ ] . pseudotyped viruses were produced as previously described [ ] . briefly, pseudotyped viruses were generated by co-transfection of dna vectors encoding hcv or vesicular stomatitis virus (vsv) envelope glycoproteins with an env-deficient hiv vector carrying a luciferase reporter gene (pnl - -luc-r --e -) into t producer cells. the plasmids used for pseudotyped virus generation have been described previously [ ] . supernatants were collected h post transfection, filtered through a . μm-pore-size filter (bd biosciences), aliquoted, frozen and subsequently titered using the for infection of cell lines, each cell line was seeded h prior to infection in -well plates such that cell confluence at the time of infection was %. triplicate wells were inoculated with hcvcc at an moi of . ffu/cell and h p.i. total cellular rna was isolated for reverse transcription followed by real-time quantitative pcr analysis (rtqpcr). for low moi hcv rna kinetic analysis, cells were seeded h prior to infection in well plates such that cell confluence at the time of infection was %. triplicate wells were infected with jfh- hcv at an moi of . ffu/cell. during the days infection assay, infected cells were trypsinized before reaching confluence and re-plated at a : dilution to maintain active growth. at indicated times p.i., medium and/or cellular rna was harvested from triplicate wells for titration or rtqpcr analysis, respectively. total intracellular rna was isolated in × nucleic acid purification lysis solution (applied biosystems, foster city, ca) and purified using an abi prism™ nucleic acid prepstation (applied biosystems), as per the manufacturer's instructions. extracellular rna from cell supernatants or intracellular hcv rna from lysed cell pellets was treated with . units of s micrococcal nuclease (fermentas, glen burnie, md) for min at room temperature, and nuclease-treated rna was isolated by the guanidine thiocyanate (gtc) method using . × gtc containing μg of murine total liver rna and following standard protocols [ ] . one μg of purified rna was used for cdna synthesis using the taq-man reverse transcription reagents (applied biosystems), followed by sybr green rtqpcr using an applied biosystems real-time thermocycler (applied biosystems). thermal cycling consisted of an initial min denaturation step at °c followed by cycles of denaturation ( s at °c) and annealing/ extension ( min at °c). hcv, human gapdh, murine gapdh, human cd , human sr-bi, human cldn , human ocln and human isg rna levels were determined relative to standard curves comprised of serial dilutions of plasmids containing the jfh- hcv cdna or the human gapdh, murine gapdh, human cd , human sr-bi, human cldn , human ocln and human isg coding sequences, respectively. the pcr primers used to amplify each respective amplicon were: universal hcv primers [ ] cells were resuspended in μl of facs buffer ( × pbs containing % (v/v) fbs, . % (w/v) nan and mm edta) and incubated for min at °c with a : dilution of antibodies specific for cd (abd serotec) or sr-bi (bd biosciences) following three rinses with facs buffer, bound antibodies were detected by incubation for h at °c with an phycoerythrin (pe)conjugated anti-mouse (bd pharmingen) antibody at a dilution of : . cells stained with an irrelevant mouse immunoglobulin g (igg) antibody and respective peconjugated secondary antibody served as negative controls. cells were washed three times, fixed in facs buffer containing % (w/v) pfa, and analyzed by flow cytometry using the dakocytomation cyan system (dako, carpinteria, ca) and summit software v . (dako). cells were fixed with % pfa (sigma, st. louis, mo) at indicated times and immunofluorescence analysis was performed as previously described [ ] . briefly, fixed cultures were rinsed three times with × pbs, permeabilized with % methanol/ % acetone (v/v) (fisher) and subsequently blocked for h with × pbs containing % (w/v) bovine serum albumin (bsa) (sigma) and % (v/v) fbs. cells were stained with a : dilution of a mouse anti-human cldn (abnova, taipei, taiwan) or mouse anti-human ocln (zymed, san francisco, ca) primary antibody overnight at °c, followed by incubation with a : dilution of an anti-mouse alexa- conjugated secondary antibody (molecular probes) for h at room temperature. cell nuclei were stained by hoechst dye. bound antibodies were visualized via confocal microscopy ( x, zeiss lsm , germany) and compared to negative control samples stained with an irrelevant mouse control antibody (santa cruz biotechnology) and alexa- conjugated secondary antibody. images were analyzed using zeiss lsm alpha imager browser v . software (zeiss), and brightness and contrast were adjusted using adobe ® photoshop ® (san jose, ca). hcv sg and fl a replicons were established as previously described [ ] . briefly, μg of in vitro-transcribed hcv genotype sg a or fl a replicon rna was transfected into cells using a modified electroporation protocol [ ] . transfected cells were diluted : , seeded in mm tissue culture dishes and maintained in the presence of g (invitrogen) at a concentration of μg/ml until all cells died or distinct g -resistant cell colonies formed. to visualize colony formation, cells were fixed with ethanol and stained with crystal violet. alternatively, colonies were expanded to establish replicon cell lines. hcvcc g was harvested from stable fl a huh replicon cells and infectivity titers were determined as described below. infectivity titration assay and immuncytochemical staining of hcv e protein culture supernatants were serially diluted -fold and used to infect -well huh cultures. at h p.i., cultures were overlayed with complete dmem containing a final concentration of . % methylcellulose (fluka bio-chemika, switzerland). seventy-two hours p.i., cells were fixed in % paraformaldehyde (sigma), and immunohistochemically stained for hcv e using a human monoclonal anti-e antibody c [ ] (a gift from drs. mansun law and dennis burton, the scripps research institute) as described [ ] . viral titers are expressed as ffu/ml, determined by the average e -positive foci number detected at the highest hcv-positive dilution. to quantify intracellular hcvcc, hcv-infected cell cultures were washed three times with × pbs, trypsinized for min at room temperature and cells resuspended in ml of % cdmem. cells were pelleted at , rpm for min at °c, frozen and then thawed three times using a % ethanol:dry ice bath. samples were then centrifuged at , rpm for min at °c to remove cell debris. the supernatant fractions were used for titration of infectious hcvcc or for hcv rna analysis, as described above. data are presented as the means ± standard deviation (sd). significant differences were determined by oneway analysis of variance (anova) followed by tukey's post hoc t test (graphpad prism © software). additional file : figure s . establishment of hepg -cd cells. (a) hepg cells, seeded in a mm tissue culture dish, were transfected with a vector control (left panel) or pee -hucd (right panel) using lipofectamine ™™ according to the manufacturer's instructions. twenty-four hours post transfection, cell culture medium was supplemented with g at μg/ml. approximately weeks post transfection, g -resistant colonies were trypsinized, pooled and cell surface expression of cd was determined by flow cytometric analysis using a mouse anti-cd antibody and an anti-mouse secondary antibody conjugated with pe. (b) cells expressing mean cd values of greater than were sorted using a becton dickinson moflo cell sorter, expanded and re-analyzed for cell surface cd expression by flow cytometry as described above (sort # ). (c) cells expressing mean cd values of greater than were sorted again, expanded and reanalyzed for cell surface cd expression by flow cytometry as described above. (sort # ) cells obtained after two rounds of cell sorting were aliquoted, frozen and designated hepg -cd cells. shaded regions represent cells stained with a monoclonal mouse control primary antibody and respective anti-mouse peconjugated secondary antibody. additional file figure s . morphological analysis of human hepatoma cell lines. (a) huh , (b) hepg -cd , (c) hep b and (d) plc cells were plated at × cells/well in a -well plate and photographed days after plating (magnification, × ). additional file figure s . hcvpp infection of different huh cell lines. huh cells lines from different laboratories [ ] were infected with equal amounts of jfhpp, h pp or vsvgpp. hcvpp entry is expressed as relative light units (rlu) ± sem for triplicate samples determined h p.i. additional file figure s . hcvcc infection of ifn-cured fl a replicon clones. plc and hep b fl a replicon cell lines replicating hcv rna at levels ≥ . × copies/μg rna were cured of hcv by co-treatment with u/ml each of ifn-β and ifn-γ for weeks. the absence of hcv rna was confirmed by rtqpcr analysis. parental huh , plc and hep b cells and cured fl a replicon clones were then infected with hcvcc at an moi of . ffu/cell. intracellular rna was collected and h p.i. and hcv rna was quantified by rtqpcr, normalized to gapdh and is displayed as hcv rna copies/μg total cellular rna (means ± sd for triplicate samples). wt = wild-type parental cells, c# = clone number. additional file figure s . hcv rna replication in huh , plc, hep b and hepg -cd cells transfected with in vitro-transcribed fulllength infectious hcv jfh- rna. huh , plc, hep b and hepg -cd cells were transfected with μg of in vitro-transcribed full-length infectious jfh- rna or a replication-deficient gnd mutant via electroporation. hcv rna was quantified by rtqpcr on indicated days post-transfection, normalized to gapdh and is displayed as mean hcv rna copies/μg total cellular rna ± sd. hepatitis c virus experimental model systems and antiviral drug research potential treatment options and future research to increase hepatitis c virus treatment response rate recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome gb virus b as a model for 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the role of transferrin receptor in hepatitis c virus entry the niemann-pick c -like cholesterol absorption receptor: a novel hepatitis c virus entry factor and potential therapeutic target egfr and epha are host factors for hepatitis c virus entry and possible targets for antiviral therapy characterization of the innate sainz et al immune signalling pathways in hepatocyte cell lines distinct poly(i-c) and virusactivated signaling pathways leading to interferon-beta production in hepatocytes hepatitis c virus induces interferon-lambda and interferonstimulated genes in primary liver cultures persistent hepatitis c virus infection in microscale primary human hepatocyte cultures modulation of hepatitis c virus rna abundance by a liver-specific microrna liver-specific microrna mir- enhances the replication of hepatitis c virus in nonhepatic cells regulation of hepatitis c virus by microrna- regulation of hepatitis c virus translation and infectious virus production by the microrna mir- micrornas in lipid metabolism the role of micrornas in cholesterol efflux and hepatic lipid metabolism interferon modulation of cellular micrornas as an antiviral mechanism single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction strand specific quantitative real-time pcr to study replication of hepatitis c virus genome the rate of hepatitis c virus infection initiation in vitro is directly related to particle density inhibition of dsrna-induced signaling in hepatitis c virus-infected cells by ns protease-dependent and -independent mechanisms enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations permissiveness of human hepatoma cell lines for hcv infection the authors graciously thank drs. lijun rong and waddah alrefai (university of illinois at chicago) for providing hep b, t and caco- cells and dr. giorgos koutsoudakis and the entire uprichard lab for helpful discussion. this work was supported by the national institutes of health public health service grants r /r -ai and r -ca and the university of illinois chicago council to support gastrointestinal and liver disease (uic gild). authors' contributions bs and slu designed the study and drafted the manuscript. bs, nb, xy and pc performed the experiments and participated in the data analysis. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -tsfxpaj authors: chai, weidong; wang, zhenya; janczyk, pawel; twardziok, sven; blohm, ulrike; osterrieder, nikolaus; burwinkel, michael title: elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (pprsv) vaccination and infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: tsfxpaj background: porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important infectious agents for the swine industry worldwide. zinc (zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. the purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with prrsv. findings: the clinical course of prrs and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet ( ppm zn, control group) and diets supplemented with zn oxide (zno) at final zn concentrations of or , ppm. pigs receiving higher dietary zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary zn levels did not substantially influence the number of antiviral ifn-gamma secreting cells (ifn-gamma-sc) or percentages of blood immune cell subsets after infection. finally, feeding higher dietary zn levels reduced neither clinical symptoms nor viral loads. conclusions: our results suggest that higher levels of dietary zno do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous prrsv infection to an extent that could improve the clinical and virological outcome. electronic supplementary material: the online version of this article (doi: . / - x- - ) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome (prrs) is one of the most significant swine diseases worldwide [ ] . efficient prrs virus (prrsv) vaccines would be invaluable in minimizing the clinical and economic impact of prrsv infections, but currently safe and effective vaccines which protect against a wide variety of strains are not available [ ] . zinc (zn) ion salts exhibit a broad-spectrum antiviral activity against a variety of viruses in vitro, including the animal viruses equine arteritis virus and transmissible gastroenteritis virus [ , ] . in the european union standard dietary zn levels in feedingstuffs are limited to ppm due to environmental reasons. however, in other countries high levels of in-feed zn oxide (zno, , - , ppm) may be added to the diet of pigs during a restricted period following weaning to prevent post-weaning diarrhea [ ] as high levels of zno have been proven to conserve the intestinal flora during the critical period following the change of diet that place at weaning [ ] . despite this effect, the exact mechanisms of zno action remain uncertain, and the local or systemic effects of zno against specific viral pathogens also remain largely unknown. we evaluated the systemic effects of different zn levels added to a conventional diet containing ppm zn (zn low , control group) against prrsv. two other groups were fed the diet supplemented with zno at final concentrations of ppm zn (zn med ), or , ppm zn (zn high ). half of the animals received a single vaccination with an experimental uv-inactivated type i prrsv (lelystad virus; lv), since it was shown that a similar vaccination with such a virus in combination with a suitable adjuvant could strongly prime the virusneutralizing (vn) response and reduce duration of viremia after homologous challenge [ ] . in contrast to vanhee et al., we chose a single-vaccination approach and challenge-infected the animals with a heterologous type i prrsv ( , % sequence identity for the envelope glycoprotein gp , which bears a major neutralizing epitope) in order test the influence of elevated zn levels on an suboptimal antigen stimulus and on crossprotection. the study was approved by the local animal welfare authority (landesamt für gesundheit und soziales, berlin, germany) under the registration number g / . german landrace piglets (n = ) of both sexes from a prrsv-free herd were weaned at the age of days, moved to a biosafety level experimental facility (bundesinstitut für risikobewertung, berlin, germany), and randomly allocated to six pens (n = per pen). piglets were assigned to three different diets ( pens per diet). at the age of days, the animals receiving the zn high diet were switched to the zn med diet, in order to avoid toxic effects of zn. one week after commencing the different diets, one pen per diet was chosen randomly and the animals were vaccinated intramuscularly with inactivated lv (accession m ; kindly provided by prof. h. nauwynck (ghent university, ghent, belgium)). four weeks after vaccination, at the age of days, all pigs were challenged with prrsv field strain cresa (accession jf ; kindly provided by prof. j. segalés and prof. e. mateu (cresa, barcelona, spain). animals were infected by intranasal application of ml of virus suspension with a titer of × tcid /ml to each nostril using a spray nebulizer. pigs were monitored daily for the presence of clinical signs and body weights were recorded weekly. blood samples were collected weekly after vaccination and at , , , , , , and days post infection (dpi). nasal swabs were taken on the same dpi as blood samples for quantification of virus shedding. all pigs were necropsied on day pi. lung and lymphoid tissues were evaluated by visual inspection for macroscopic lesions, and samples from lungs, lymph nodes, tonsils, and spleen were taken and immediately frozen in liquid nitrogen and stored at − °c. for virological analysis, serum samples ( , , , , dpi), nasal swabs ( , , dpi), and tonsil, lung and tracheobronchial lymph node samples ( dpi) were examined by qpcr to determine prrsv copy numbers. rna extraction was performed using a viral rna/dna purification kit (stratec) applying μl of serum or mg of tissue each. rna yields and quality were determined with a nanodrop® nd- spectrophotometer (nanodrop technologies). reverse transcription (rt) was performed using the dy-namo™ cdna synthesis kit (thermo fisher scientific). viral loads were quantified using a taqman fluorescent probe-based real-time qpcr assay in an icycler iq™ detection system (bio-rad) with primers described elsewhere [ ] . prrsv-specific igm and igg antibodies were measured by elisa (ingezim prrs dr, ingenasa) according to the manufacturer's instructions. vn antibodies against prrsv were quantified by a viral neutralization test as previously described [ ] . neutralization of prrsv strain cresa was examined using prrsv gp specific monoclonal antibody h (ingenasa) and alexa fluor™ conjugated anti-mouse igg (h + l) secondary antibody (invitrogen). peripheral blood mononuclear cells (pbmc) were isolated using density centrifugation through a ficoll gradient (lsm , paa laboratories). samples were treated with erythrocyte lysis buffer for min on ice, pbmc were washed with ml of pbs with . % bsa and centrifuged for min at × g at °c. in all samples, pbmc viability was confirmed using standard procedures. the cell-mediated prrsv-specific immune response was measured by using elispot for the enumeration of ifn-gamma-sc in pbmc (mabtech). in order to compare homologous and heterologous responses, pbmc were stimulated in parallel ( . × pbmc/well, wells per pig and stimulus) with cresa or lv at a multiplicity of infection of . . unstimulated and phastimulated cells ( μg/ml) were used as negative and positive controls, respectively. ifn-gamma-sc numbers were counted using an elispot reader system (a.el. vis gmbh). flow cytometry analysis was performed as described before [ ] using a bd facscanto™ flow cytometer (bd biosciences). data were analyzed with flowjo™ software (treestar). almost all infected pigs showed clinical symptoms typical for prrsv infection and similar to a previous study with the same prrsv strain [ ] . increased rectal temperatures were detected for more than days, and edema of the eyelids and conjunctivitis for more than days. there was no significant dietary effect on fever magnitude and duration (figure a,b) . other clinical signs such as cough were observed only sporadically and lasted only for - days. regarding the weight gain, we could only analyze the effect of the zn med diet for the vaccinated groups, given that time points were missing for the pigs receiving the zn high pigs. for the remaining time points as well as for the non-vaccinated groups we found no benefits of feeding higher dietary zn levels (figure c,d) . mean prrsv load in serum as determined by qpcr peaked at dpi and gradually reduced later on. neither vaccination nor elevated zn levels showed an influence on prrsv viremia at any time during the observation period (figure a,b) . the same was observed regarding virus shedding as determined by analysis of nasal swabs (figure c,d) . persistent virus was found at dpi in the majority of tonsil samples, regardless of diet, while all lung and tracheobronchial lymph node samples were tested negative for the presence of prrsv genomes. prrsv-specific igg and igm antibodies were not detected by elisa in randomly chosen samples ( vaccinated/ non-vaccinated) before infection on day before infection. on day , only one vaccinated animal was positive for prrsv antibodies (figure a,b) . this is in contrast with previous results showing earlier seroconversion after vaccination with uv-inactivated lv [ ] and might be due to the fact that we used a cell culture-adapted non-purified lv with possibly decreased immunogenicity compared to purified lv grown on porcine alveolar macrophages used in the cited study. at dpi, piglets from all groups were seropositive, except for pig each in the vaccinated and non-vaccinated zn low groups. higher (p ≤ . ) antibody levels were detected in vaccinated groups than in non-vaccinated groups at and dpi after prrsv challenge, while the diet had no influence on antibody levels. the generation of neutralizing antibodies is delayed in prrsv infection and usually appears three to four weeks after infection [ ] . accordingly, in our study virus neutralizing vn antibodies were not detectable until dpi in the serum. a single-vaccination with an inactivated lv did not lead to an earlier induction of vn antibodies, but vaccinated groups developed higher (p = . ) vn antibody titers than their non-vaccinated counterparts at dpi ( figure c, d) . a tendency towards higher vn antibody levels was evident in pigs receiving higher levels of zn (zn med ) at dpi. this tendency continued to dpi in both zn med and zn high groups. thus, the possibility remains that animals receiving higher dietary zn levels might be better protected against reinfection with prrsv. the number of ifn-gamma-sc at dpi revealed no differences after homologous or heterologous re-stimulation (additional file ). flow cytometry analysis of pbmc phenotypes was performed weekly from day to day post infection. single vaccination only delayed but not prevented the prrsv-induced decrease of cd + , cd + and cd + cd + t cell populations, which are important for viral clearance [ ] . we found no sustained effect of dietary zn levels on any of the analysed cell subsets (additional file ). necropsies at dpi revealed no gross lung lesions and lymphoid hyperplasia in tonsils, lymph nodes or spleen in any of the pigs. overall, the study shows that challenge infection with a wild-type prrsv without additional environmental and social stress and the impact of secondary infections results in relatively mild clinical signs. under these conditions, elevated dietary zn levels could not provide enhanced protection against infection with a type i prrsv field strain and could not improve efficacy after a singlevaccination with a heterologous inactivated vaccine. the data sets supporting the results of this article are included within the article (and its additional files). additional file : figure s . prrsv-specific numbers of ifn-gamma-sc determined by elispot. pbmc collected at dpi were restimulated with either of the prrsv strains (lv or cresa ) used in the study. results are shown as average frequencies of virus-specific ifn-gamma-sc per . × pbmc. filled symbols indicate results obtained after in vitro restimulation with the same prrsv used for infections (homologous) while empty symbols show the results of in vitro restimulation with lv (heterologous). additional file : figure s . modulation of pbmc immune cells frequencies determined by flow cytometry analysis. a and b, cytotoxic lymphocytes (cd + cd − cd α high ); c and d, naïve t h cells (cd + cd + cd − ); e and f, cd + γδ t cells (cd + cd + cd + ); g and h, antibody forming and/ or memory b cells (cd − cd + cd − ); i and j, nk cells. asterisks indicate statistically significant differences (p < . ) between averages at each dpi. immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology. vaccine zn( +) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture antiviral activity of zinc salts against transmissible gastroenteritis virus in vitro effect and interaction between wheat bran and zinc oxide on productive performance and intestinal health in post-weaning piglets dietary zinc oxide in weaned pigs-effects on perfor-mance, tissue concentrations, morphology, neutrophil functions and fecal microflora development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant dietary enterococcus faecium ncimb and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection the challenge of prrs immunology evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (pprsv) vaccination and infection the authors would like to acknowledge the animal welfare officer m. ladwig and all animal technicians supervised by dr. s. banneke at bundesinstitut für risikobewertung for their engagement. we further acknowledge e. luge for his excellent technical assistance. we thank dr. s. kreuzer, züchtungsbiologie und molekulare genetik, humboldt-universität zu berlin, for helping with the flow cytometry data analysis and prof. m. schmidt, institut für immunologie, freie universität berlin, for helpful comments about the experimental design. we also thank b. esch, institut für virologie, freie universität berlin for expert technical assistance. the study was funded by the deutsche forschungsgemeinschaft through grant sfb / . the authors declare that they have no competing interests. key: cord- - ygcfzii authors: mehrbod, parvaneh; eybpoosh, sana; fotouhi, fatemeh; shokouhi targhi, hadiseh; mazaheri, vahideh; farahmand, behrokh title: association of ifitm rs polymorphisms, bmi, diabetes, and hypercholesterolemia with mild flu in an iranian population date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: ygcfzii background: ifitm has been suggested to be associated with infection in some ethnic groups. diabetes and hypercholesterolemia are also important clinical conditions that can predispose individuals to infection. the aim of this study was to investigate the association of rs c polymorphism, bmi, diabetes, and hypercholesterolemia with mild flu in an iranian population. methods: we conducted a case-control study, including mild flu and flu-negative individuals attending primary care centers of three provinces of iran (i.e, markazi, semnan, and zanjan). pharyngeal swab specimens were collected from all participants, and were subjected to rna and dna extractions for real-time pcr and pcr tests. all pcr products were then sequenced to find t/c polymorphisms in the rs region. data on demographic, anthropometric, and clinical variables were collected from participants’ medical records available in the primary care centers. the data was analyzed using dnasis (v. . ) and stata (v. ) software. results: all participants were of fars ethnic background. the allele frequency for rs -c was found to be . % among cases and . % among controls. carriers of the rs c allele (ct + cc genotypes) showed . folds increase in the risk of mild flu comparing to the t allele homozygotes (p value: . ). we also found a significant positive association between rs c allele heterozygote and mild flu (or: . , p value: . ), but not in c allele homozygote group (or: . , p value: . ). similarly, we did not find a significant association between mild flu and bmi (or: . , p value: . ), diabetes (or: . , p value: . ), and hypercholesterolemia (or: . , p value: . ) in multivariable logistic regression. conclusions: this is the first study evaluating the association between rs polymorphisms, diabetes, hypercholesterolemia, and bmi and susceptibility to mild flu in an iranian population. our results suggest a significant positive association between mild flu and rs c allele heterozygous and carriage. future replication of the strong association observed here between rs c allele carriage and mild flu might candidate this polymorphism as a genetic marker for early screening of susceptibility to mild flu. lack of significant association between c allele homozygous and mild flu, observed in this study, might be the result of small sample size in this group. trial registration: ir.pii.rec. . . electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. influenza is the result of both host and viral genetic components combination. the viral genetic determinants and host immunity have been widely considered, while the host genetic determinants remain elusive [ ] . the study of the host genetic factors involved in susceptibility to influenza is a promising strategy that may identify potential therapeutic targets [ ] . as a result, systematic investigation of the association between host genetic factors and the occurrence of influenza is of pivotal importance. in , the world health organization (who) identified as priority the studies which considered the role of host genetic factors on susceptibility to severe flu [ ] . during viral infection, viral proteins and viral nucleic acids are detected by pathogen recognition receptors (prrs). these recognition patterns activate signaling transduction pathways that trigger production of type i interferon and other related cytokines [ , ] . type i interferon then elevates the expression of hundreds of genes, named interferon stimulated genes (isgs), that weaken virus replication by a variety of mechanisms [ , ] . it has been suggested that the expression of interferoninducible transmembrane (ifitm) genes restrict the replication of several highly pathogenic human viruses, especially those that enter the cell via acidic endosome [ , , ] . these viruses include severe acute respiratory syndrome (sars), coronavirus, marburg and ebola viruses, influenza a viruses (iavs), dengue virus, and hiv- [ , , , , ] . in humans, it has been predicted that rare snp rs -c allele of ifitm produces an alternatively spliced transcript that encodes a truncated protein (Δ ifitm ) leading to reduced control of virus replication in vitro. however, expression at mrna or protein level of the Δ ifitm variant has not been detected to date [ , ] . the value of ifitm in influenza virus infection was determined by everitt et al. who showed that severity of influenza virus infection was greatly increased in ifitm knockout mice compared to the wild-type animals [ ] . everitt et al. and zhang et al. demonstrated increased morbidity and mortality in rs -c homozygotes during the h n influenza pandemic. due to the high frequency of the rs -c allele in asians, the effect of the homozygosity for this allele was proposed to translate to a population-attributable risk of . % for severe flu in the chinese population compared with . % in northern europeans. both studies were performed with small sample sizes of h n -infected patients, calling for further studies on large sample sizes. two studies reported a strong association between ifitm snp rs -c and worse clinical outcomes in patients infected with iav pandemic. the studies, however, doubt the presence of such association, at least among europeans, as several rs- , -c homozygous patients only developed a mild flu [ , ] . it appears that ifitm inhibits an early event after endocytosis of virion particles, by blocking virus release into the cytosol [ , , ] . ifitm protein is considered to be the most active against iav and resides in the late endosomes and lysosomes using several orthologous genetic approaches [ , ] . wang and colleagues showed that inflammatory immune responses linked to the c/c ifitm genotype during h n infection play an important role in the pathogenesis of influenza. they found that patients with rs -c/c ifn-ifitm genotype had more rapid disease progression, and were less likely to survive [ ] . although previous studies concluded that the rs -c variant leads to reduced control of virus replication in vitro, posterior studies did not find such effects. for example, mills et al. found an association between rs rare allele c/c homozygotes and susceptibility to mild flu but they could not confirm previous reports for the association between this polymorphism and susceptibility to severe h n infection [ ] . the data obtained by lópez-rodríguez et al. did not either suggest any role of rs -c in the development of severe iav in their studied population. they suggested these groups might be at risk of severe influenza, hence individualized measures in the case of iav is required [ ] . giao et al. also showed that except h n influenza (pdm ), the risk of hospitalization due to other respiratory infections was higher among rs c allele carriers comparing to patients with tt genotype [ ] . it has been reported that local accumulation of cholesterol to the plasma membrane is important for clustering of viral structural proteins in lipid rafts to help assemble the virus particles [ ] . thus, the imbalance in cholesterol level may play a role in the susceptibility to influenza. the ifitm gene mutation may lead to an increase in impaired cholesterol levels [ ] . as an instance, mutation rate of ifitm gene is shown to be high among han chinese population. coronary artery disease and ischemic stroke are highly associated with bmi and serum cholesterol levels, and are the leading causes of morbidity and mortality in han chinese population [ , , ] . therefore, we performed this study to identify the association between mild flu and ifitm rs -c polymorphism, bmi, diabetes and hypercholesterolemia. written informed consent was obtained from all participants. all procedures performed in this study involving human participants were according to the principles helsinki declaration. the research protocol was approved by the ethics committee of pasteur institute of iran (ethics code: ir.pii.rec. . ). we performed a case-control study on the samples collected from march to december in three provinces of northern-central iran, including markazi, semnan, and zanjan. cases would be included in this study if they had apparent symptoms of respiratory tract infection, and were confirmed as mild flu with pcr test. controls were flu-negative individuals based on the pcr results, and would be included in the study if they had no history of influenza. both cases and controls should be based and currently residing in markazi, semnan, or zanjan provinces. having respiratory comorbidities, consumption of immune suppressive drugs, and previous vaccination against influenza virus were our exclusion criteria. demographic and anthropometric characteristics measured for each participant included participants' age (in years), province (markazi, semnan, and zanjan provinces), body mass index (bmi, kg/m ), diabetes (yes/no), hypercholesterolemia (yes/no), and genotype in the ifitm rs locus. the bmi of each participant was calculated using the formula: bmi = [weight (kg)] / [height (m)] . the diabetes and hypercholesterolemia positive and negative cases were confirmed based on the patients' history reports. to test for influenza, pharyngeal specimen from each individual was obtained with a dacron swab. specimens were collected on dry ice in transport media containing pen/strep and amphotericin b, and then were transferred to pasteur institute of iran for determination of iav. all samples were stored at °c for h. following vortex, swabs were discarded and the supernatants were aliquoted in sterile labeled micro-tubes and stored at − °c for further testing. for each sample, viral rna was extracted from μl fluid specimen using high pure viral rna kit, according to the manufacturer's instructions (roche, switzerland). the extracted rna was isolated and resuspended in μl elution buffer and stored at − °c for real-time pcr assay. in quantitative real-time pcr (qrt-pcr), primers and probes were designed and synthesized by sinaclon co. (iran) based on the latest who guideline. table describes the primers used for amplification of viral genes and rnasep as housekeeping control. real-time pcr reactions were performed in total volume of μl, using rotor-gene q (qiagen). the reaction mixture consisted of x master mix, superscript iii rt/ platinum taq mix, specific primers and fluorescent probes. the thermal cycling program was followed based on who guideline. briefly, reverse transcription was carried out at °c for min, followed by °for min taq inhibitor activation, and amplification at °c for s followed by °c for s. human dna extraction for each sample, genomic dna was extracted from μl fluid specimen using dnp™ high yield dna purification kit, according to the manufacturer's instructions (cinnagen, iran). briefly, μl of lysis solution was added to each sample. following vortex and addition of precipitation solution, a second round vortex was performed. the samples were then centrifuged. wash buffer was used for two times to wash the precipitation. finally, solvent buffer was added and the tubes taqman probes are labeled at ′-end with the reporter molecule carboxyfluorescein (fam) and quenched internally at a modified "t" residue with bhq with a modified ′-end to prevent probe extension by taq polymerase were warmed at °c for min. the supernatant, which was the purified dna, was stored at − °c for pcr assay. primers for pcr were designed and synthesized by sinaclon co. (iran) based on previous published articles [ ] . the primers used for the amplification of human ifitm rs were as follow: ifitm -f ′-gg aaactgttgagaaaccgaa- ′ and ifitm -r ′-catacgcaccttcacggagt- ′ to produce bp pcr product. the s rrna was amplified as internal housekeeping using theses primers sequences: s-f ′-ggccctgtaattggaatgagtc- ′ and s-r ′-ccaagatccaactacgagctt- ′ to produce bp pcr product. pcr reaction was performed in total volume of μl, using thermocycler (germany). the reaction mixture consisted of taq dna polymerase × master mix red with . mm mgcl (amplicon, denmark), specific primers and dnase-free water. amplification was carried out at °c for s in order to pre-denature the template dna, followed by cycles of denaturation at °c for s, annealing at °c for min and extension at °c for min. the final extension was completed at °c for min. the pcr products were purified using pcr purification kit (millipore), and subjected to sequencing (first base co, malaysia) using the bigdye® terminator v . cycle sequencing kit chemistry. then they were loaded into dna analyzer (gatc biotech). both sense and antisense strands of pcr products were directly sequenced using the same primers as the pcr amplification. snps were detected by direct sequence analysis using dnasis max. software. the reference sequence for the ifitm gene was based on the sequence of human chromosome , clone grch .p . hardy-weinberg equilibrium (hwe) was tested in cases and controls using the web tool developed by rodriguez, and colleagues [ ] . using this tool, allele frequency and genotype distribution were calculated for cases and controls, as well. statistical analyses were performed using stata software (version ). continuous and categorical data were described in the usual fashion; means ± sds were used to describe continuous variables, and numbers (percentages) were used to describe categorical variables. association between categorical variables, including study group (mild flu vs. flu-negatives), rs genotype (cc, ct, tt, and c carriers), rs allele (c and t), province (markazi, semnan, and zanjan), diabetes (positive vs. negative), and hypercholesterolemia (positive vs. negative), was assessed using pearson's chi-squared test. for each cell with zero counts in contingency tables, yates's correction was used. also, where more than % of cells had counts less than , the alternative fisher's exact test was used instead of pearson's chi-squared test. univariable logistic regression was used to test the association of each independent variable with mild flu. variables with a p value smaller than . in the univariable model were included into the multivariable logistic regression. also, a backward method using likelihood ratio tests (lrt) was used to make the model as simple as possible. as we sampled cases and controls from three distinct provinces of iran, we retained the variable "province" in the final model. this was to ensure that residual population admixture, due to differences in participants' area of residence, does not confound the association between rs genotypes and mild flu. all statistical tests were two-tailed with a type i error of . . observed powers of the estimated crude and adjusted odds ratios (or) were also calculated to assess the robustness of our results (additional file ). a total of mild flu cases and controls were included in this study. participants' mean age in the case and control groups was . (sd: . ) and . (sd: . ) years, respectively (p value t-test : . ). all participants were of fars ethnicity, which is the dominant ethnicity in iran. the distribution pattern of flupositive and flu-negative individuals was similar across three provinces, with semnan having the highest number of both cases ( . %) and controls ( . %), and zanjan having the lowest number of cases ( . %) and controls ( . %). the difference in the number of cases and controls was significant between semnan and zanjan (or seman vs. zanjan : . , p value: . ), but not between semanan and markazi or between zanjan and markazi provinces. details of participants' characteristics are shown in table . the mean bmi value was slightly higher among mild flu patients rather than in controls (p value: . ). prevalence of hypercholesterolemia and diabetes was also significantly higher in controls rather than in mild flu patients (p values: . and . , respectively; table ). the tt genotype was the most prevalent genotype both among cases ( . %) and controls ( . %). similarly, the cc genotype was the least prevalent genotype in both groups (cases: . %, controls: . %, table ). in both groups, the alleles were in hwe (p value > . , table ). the distribution of tt, tc, cc genotypes as well as c carriers was not significantly different across three provinces, either in total, or separately among cases and controls ( table ) . results of the univariable logistic regression showed that having tc genotype rather than tt, significantly increased the risk of mild flu (or: . , p value: . ). the same effect, but with greater magnitude, was also observed for the cc genotype rather than the tt genotype, however, this effect was not statistically significant (or: . , p value: . ; table ). in the final model, the effect of both tc and cc genotypes on the risk of mild flu showed an increase, but again this effect was only significant in the tc group. furthermore, carriers of the rs c allele (ct + cc genotypes) showed . folds increase in the risk of mild flu comparing to the t allele homozygotes (p value: . ). in the final model, the effect of bmi, diabetes, and hypercholesterolemia were no longer statistically significant ( table ) . details of multivariable regression process are presented in additional file . advances in the genomic area have enlightened the role of genetic factors in susceptibility to various infectious diseases. in this regard, ifitm protein encoded by ifitm gene, have shown to be associated with susceptibility to several viral infections, such as west nile virus, dengue virus, rhinovirus, corona virus, hiv, respiratory syncytial virus, and influenza a virus [ , , , ] . in case of influenza infection, however, it is not clear whether the reported association relates to the infection severity. in this study, we investigated the association between mild flu and ifitm rs variant, bmi, diabetes, and hypercholesterolemia in an iranian population. our results suggest a significant positive association between mild flu and rs c allele heterozygous (ct) and carriage (ct + cc), but not between mild flu and c allele homozygous (cc), bmi, diabetes, and hypercholesterolemia. ifitm/ ifitm proteins are membrane-associated proteins, and directly interfere with the virus entry into the host's cells [ ] . since virus entry often proceeds through endocytosis, it is possible that ifitm proteins are involved in endocytosis, by detecting and eliminating viral invaders before establishment of infection [ , ] . therefore, mutation in the gene coding for these proteins can lead to susceptibility to various infections. yount, et al. ( ) introduced ifitm in mouse dendritic cells. they mentioned s-palmitoylation of ifitm on proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. [ ] . bailey and colleagues also found that iav lung titers were higher in ifitm knockout mice versus wild-type animals [ ] . the association of ifitm gene polymorphisms with mild and sever influenza has been investigated in different ethnicities. for example, zhang, et al. pointed to the significant role of ifitm genetic variants on the epidemiology of influenza among chinese individuals [ ] . yang, et al. found a significant association between ifitm -snp rs and susceptibility to severe, but not mild flu, among asian and caucasian ethnicities [ ] . conversely, mills, et al. found evidence of an association between rs c allele homozygotes and susceptibility to mild flu in patients with caucasian ancestry, attending primary care, but could not confirm the association between this snp and susceptibility to severe flu [ ] . studies conducted by lópez-rodríguez and giao, et al. did not find evidence for the association between rs c allele homozygotes and susceptibility to sever flu [ , , ] . based on the conclusion from the review paper of ciancanelli et al. which mentioned ifitm snp could not be a major causal factor, but perhaps a modifier, of the outcome of influenza infection [ ] , we also concluded there is only convincing experimental evidence for the role of wild-type ifitm but further work is required to define the role of ifitm snps as modifiers for severe influenza. the inconsistent results reported by studies investigated the association between ifitm snp rs and influenza, made yang, et al. to conduct a systematic review. their meta-analysis included patients with influenza and controls from four studies, including different ethnic and severity subgroups. their results showed that patients with the rs -c/c genotype were more likely to increase the susceptibility to severe but not in mild flu, in both uk caucasians and han chinese [ ] . our results also showed that rs -c carriers have an increased risk of mild flu rather than patients with tt genotype. this may be explained as due to the differences in participants' ethnic background, since we studied iranian ethnicities while yang, et al. focused on uk caucasians and han chinese ethnicities. although, the rs -c allele in the iranian population was rare, the overall effect of this allele on the risk of influenza was found to be considerably high. as our sample size was small, the power for some of our estimates was not desirable. in case such findings being replicated by future studies, the knowledge can be used in effective management of the epidemics. for example, understanding the biological pathways leading to mild and severe flu may lead to improvement of therapeutic options, and can alter existing vaccination practices. findings of largescale studies on genetic susceptibility to influenza would also guide population-based prevention efforts. for example, by considering the genetic susceptibility of different sub-populations to influenza virus in a country, human and medical resources would be allocated to high risk areas in case epidemics/pandemics occur. the ifitm allele frequency varies significantly among different ethnicities. for example, the frequency of rs c allele homozygotes in han chinese, japanese, northern europeans, and englanders has been shown to vary greatly, from % among japanese to % among chinese populations ( , , and %, respectively) [ ] . little is known about the frequency of this genotype in iran. in our study, the c allele frequency observed among flu-negative individuals was . %, resembling the allele frequency among englanders. studies with larger sample sizes would provide a better understanding about the frequency of susceptibility alleles in iran. in this study, we recruited our samples from three provinces of northern-central iran, including markazi, semnan, and zanjan provinces. although all participants were of iranian ethnicity, participants of each province might be different regarding their genetic and social history, leading to residual confounding due to population stratification. population stratification is related to situations when cases and controls are of different ethnic backgrounds with different genetic compositions, and has been noted as the most important type of the bias in genetic association studies. to control for the effect of residual population admixture on our results, we adjusted genetic association between ifitm -snp rs and susceptibility to mild flu for participants' residence area. growing empirical and theoretical evidence suggests that statistical adjustment for confounding effect of ethnicity provides estimates of genetic associations which are robust to bias from population stratification [ ] . in our study, we did not observe a significant association between rs c allele homozygote, bmi, diabetes, and hypercholesterolemia and mild flu. this might be due to relatively small sample size in our study, which led to moderate power of some parameter estimates. to verify these findings, further studies on the iranian ethnicity with larger sample sizes are recommended. to the best of our knowledge, this is the first study evaluating the association between ifitm rs polymorphism, diabetes, hypercholesterolemia and bmi with susceptibility to mild flu in an iranian sample. our results suggest a significant positive association between mild flu and rs c allele heterozygous and carriage. however, we did not observe an association between mild flu and rs c allele homozygous, bmi, diabetes, and hypercholesterolemia. further studies are recommended to verify these findings in a larger sample of iranian individuals. future replication of the strong association observed here between rs c allele carriage and mild flu would be beneficial in candidating this polymorphism as a genetic marker for early screening of susceptibility to mild flu. the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry ifitm limits the severity of acute influenza in mice ifitm proteins mediate the innate immune response to influenza a h n virus, west nile virus and dengue virus population stratification and spurious allelic association characteristics of ifitm, the newly identified ifninducible anti-hiv- family proteins host genetics of severe influenza: from mouse mx to human irf natural mutations in ifitm modulate post-translational regulation and toggle antiviral specificity serum pcsk is associated with multiple metabolic factors in a large han chinese population ifitm restricts the morbidity and mortality associated with influenza ifitm inhibits influenza a virus infection by preventing cytosolic entry hospitalization risk due to respiratory illness associated with genetic variation at ifitm in patients with influenza a(h n )pdm infection: a case-control study major causes of death among men and women in china the role of host genetics in susceptibility to influenza: a systematic review distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections mutations in human genes that increase the risk for severe influenza infection microb independent human host factors required for influenza virus replication initial impact of the sequencing of the human genome ifitm and severe influenza virus infection. no evidence of genetic association the ifitm proteins inhibit hiv- infection virus entry by endocytosis ifitm and susceptibility to respiratory viral infections in the community single nucleotide polymorphisms in the proximal promoter region of apolipoprotein m gene (apom) confer the susceptibility to development of type diabetes in han chinese hardy-weinberg equilibrium testing of biological ascertainment for mendelian randomization studies interferon-inducible antiviral effectors a diverse range of gene products are effectors of the type i interferon antiviral response early hypercytokinemia is associated with interferon-induced transmembrane protein- dysfunction and predictive of fatal h n infection interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms interferon-inducible transmembrane protein genetic variant rs and influenza susceptibility and severity: a meta-analysis palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm interferon-induced transmembrane protein- genetic variant rs -c is associated with severe influenza in chinese individuals the authors would like to thank dr. amin doosti irani for his valuable consultation in statistical analysis. this study was funded by national elites foundation, iran (grant number ). all data in case of need are available. written informed consent was obtained from the patients or the guardians of the patients participated in this research. submit your next manuscript to biomed central and we will help you at every step: key: cord- -oe onyr authors: vasilakis, nikos; guzman, hilda; firth, cadhla; forrester, naomi l; widen, steven g; wood, thomas g; rossi, shannan l; ghedin, elodie; popov, vsevolov; blasdell, kim r; walker, peter j; tesh, robert b title: mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: oe onyr background: the family mesoniviridae (order nidovirales) comprises of a group of positive-sense, single-stranded rna ([+]ssrna) viruses isolated from mosquitoes. findings: thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in indonesia, thailand and the usa. by electron microscopy, the virions appeared as spherical particles with a diameter of ~ nm. their , nt to , nt genomes consist of positive-sense, single-stranded rna with a poly-a tail. four isolates from houston, texas, and one isolate from java, indonesia, were identified as variants of the species alphamesonivirus- which also includes nam dinh virus (ndiv) from vietnam and cavally virus (cavv) from côte d’ivoire. the eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: karang sari virus (ksav) from java, bontag baru virus (bbav) from java and kalimantan, and kamphaeng phet virus (kphv) from thailand. in comparison with ndiv, the three new mesoniviruses each contained a long insertion ( – nt) of unknown function in the ’ region of orf a, which accounted for much of the difference in genome size. the insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. conclusions: in summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family mesoniviridae, order nidovirales. species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as ndiv and cavv (alphamesonivirus- ) and the other eight isolates would represent three new mesonivirus species (alphamesonivirus- , alphamesonivirus- and alphamesonivirus- ). the observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide. the recently established virus family mesoniviridae (order nidovirales) comprises a group of positive-sense, singlestranded rna ([+]ssrna) insect viruses [ ] . to date, the six described mesoniviruses, cavally (cavv), daknong (dkng), hana (hanav), meno (menov), nam dinh (ndiv) and nse (nsev) have all been isolated from naturally infected mosquitoes collected in just two countries: côte d'ivoire (west africa) and vietnam (southeast asia) [ ] [ ] [ ] [ ] . although these mosquito-associated viruses do not appear to infect vertebrates or to cause illness in humans or livestock, they are nonetheless of interest because of the structural and genetic similarities to other members of the order nidovirales, namely viruses in the families coronaviridae, arteriviridae, and roniviridae. furthermore, the basal phylogenetic position of the mesoniviruses in relation to the coronaviridae has led some authors to suggest that the coronaviruses, and possibly other viruses in the order nidovirales, may have evolved in arthropods [ ] [ ] [ ] . indeed, members of the roniviridae naturally infect marine shrimp and can cause severe pathology in these economically important arthropods [ ] . in this communication, we report the isolation and characterization of additional mesoniviruses from mosquitoes collected in thailand, indonesia and the united states of america (usa). these viruses appear to represent four distinct species in the family mesoniviridae, three of which are novel. based on their wide geographic distribution, the limited sampling that has been done to date for these mosquito-specific viruses, and their broad species host range in mosquitoes, it seems likely that mesoniviruses are common in mosquito populations worldwide. the potential biological significance and effect of mesoniviruses on mosquito vector competence is also discussed. all isolates had similar ultrastructure. mature virions nm in diameter were located at the surface of infected c / cells, either as individual particles or in groups ( figure b,d) , similar to what has been reported recently [ ] . they displayed a dense nucleocapsid core~ nm in diameter and surrounding envelope ( figure a ,b). mature virions of the same size could also be found inside intracytoplasmic vacuoles ( figure a ,b,d,e,f,h). some infected cells had paracrystalline arrays consisting of empty and full virus particles but with less electron-density than mature virions ( figure c ). at the periphery of these arrays, mature virions could be observed either free in the cytosol or inside vacuoles ( figure c ). moreover a recent report [ ] , suggests that the mesonivirus virion diameter of~ - nm observed by us and others [ ] may be significantly lower than its actual size, a discrepancy attributed to the method of preparation for alternative imaging technologies. employing cryoelectron tomography, warrilow et al. [ ] demonstrated on the surface of some virions the presence of spikelike projections displaying a globular head attached to the virion surface through a low density stalk, similar to what has been observed in other members of the order nidovirales [ ] . the complete nucleotide sequences of all isolates were determined by high-throughput illumina sequencing with end-finishing by '-and '-race. excluding the '-poly [a] tail, the (+) ssrna genomes ranged in size from , nt (v ) to , nt (jkt- ). the organization of each genome was similar to that described previously for the mesoniviruses (ndiv, cavv, hanav, nsev and menov), featuring a long '-untranslated region ( '-utr) of to nt, six major long open reading frames (orfs), and a long terminal region of to nt preceding the poly[a] tail ( figure ). an alignment that also included the five previously described mesoniviruses revealed block insertions in three groups of isolates: i) kp - , kp - and kp - had a nt insertion, ii) jkt- , jkt- , jkt- and jkt- had a nt insertion, and iii) jkt- had a nt insertion. these insertions accounted for most of the observed difference in genome size in the mesoniviruses ( figure ). excluding this region, which commenced~ nt from the 'terminus, the mesoniviruses shared global nucleotide sequence identity of~ . %. maximum pairwise divergence was between menov and all other viruses ( . % - . % identity). to determine the phylogenetic relationships of the newly identified insect viruses, maximum likelihood (ml) phylogenetic trees were constructed based on the amino acid alignments of orf a (unprocessed s protein) and a concatenated region of the highly conserved domains within orf ab ( cl pro , rdrp and znhel ). the phylogenies exhibited highly similar topologies and strongly indicated that the viruses identified in this study cluster within the previously identified insect-specific nidoviruses in the genus mesonivirus (ndiv, cavv, hanav, nsev and menov) ( figure ) . a multiple sequence alignment of the new genomes indicated high identity between multiple isolates, which was also confirmed by our phylogenetic analysis. isolates kp - , kp - and kp - differed only by ten nucleotide substitutions and a single -nt indel and formed a well-supported monophyletic clade in both phylogenies ( figure ). these should be considered isolates of the same virus (designated kamphang figure genome organisation of the mesoniviruses. in orf a and orf b the putative ribosomal frame-shift (rfs) element and the locations of conserved functional domains, including the c-like serine protease ( clp), rna-dependent rna polymerase (rdrp), zinc-binding (z), helicase (hel), exoribonuclease (exon), n -methyltransferase (nmt) and '-o-methyltransferase (omt) domains, are shown. sequence insertions in orf a are shown as black blocks. in orf a encoding the s glycoprotein the sites of proteolytic cleavage to generate s , s and the uncharacterised n-terminal fragment are shown as black inverted triangles. putative orf that is relatively conserved in the 'terminal region of the genome is shown as black rectangles and other small orfs > nt that occur variously in the '-terminal domains and elsewhere in the genome are shown as grey rectangles. phet virus -kphv). likewise, jkt- , jkt- and jkt- differed by only single nucleotide substitutions and jkt- differed by nucleotide substitutions and two indels of and nucleotides, and these four viruses formed a strongly supported monophyletic clade in the ml trees, suggesting that they can be considered isolates of the same virus (designated botang baru virus -bbav). isolates , , v and v were also closely related, differing by only - nucleotide substitutions (~ . - . %) across the entire genome. the clade formed by these isolates in each ml tree clustered within a closely related monophyletic group that also included ndiv and isolate jkt- ( figure ). these can all be considered strains of ndiv. finally, isolate jkt- was clearly distinct from all other mesoniviruses in our phylogenetic analysis, and was considered a unique virus, karang sari (ksav). it has recently been suggested that pairwise evolutionary distances (ped) can be used as a species demarcation criterion in the genus mesonivirus [ ] . we calculated the ped between the new isolates described in this study and those of five previously described mesoniviruses (cavv, hanav, ndiv, nsev and menov) using the conserved protein domains of orf ab ( cl pro , ). based on this analysis, ndiv (including the ngewontan and houston strains) and cavv can be considered a single species, which has previously been designated at alphamesonivirus- . the ped between each of these strains was substantially less than the suggested cutoff of . for species demarcation [ ] . by this method, each of the other previously described mesoniviruses -hanav menov, and nsevwould also be considered unique species, as the ped between these viruses and all other mesoniviruses were considerably greater than . . these viruses could be assigned to the species alphamesonivirus- , alphamesonivirus- and alphamesonivirus- , respectively. similarly, novel viruses bbav, ksav and kphv would each also be assigned to separate mesonovirus species: alphamesonivirus- , alphamesonivirus- and alphamesonivirus- , respectively. strikingly, the vast majority of the genetic diversity that was measured within the genus mesonivirus was present at the inter-species level (additional file : figure s ). this pattern strongly supports the assignment of the current diversity of mesoniviruses into seven distinct species. in mesoniviruses, orf a features a c-like protease ( clpro) domain and flanking transmembrane domains, which are highly conserved amongst nidoviruses; these domains were identified in each of the new viruses. however, the block nucleotide sequence insertions resulted in a highly variable region near the n-terminus of orf a with insertions of various lengths ranging from aa (menv) to aa (ksav) (additional file : figure s ). the inserted sequences (which apparently are unique to the mesoniviruses) featured variations on a common sequence (skrkgk) at the terminal points. there were also various short stretches of this sequence and other imperfectly repeated sequences within each insertion region suggesting possible origins through recombination or sequence duplication events. as has been reported previously for other mesoniviruses, orf a is overlapped by orf b which contains highly conserved regions associated with the replicase complex including rna-dependent rna polymerase (rdrp), multinucleate zinc-binding module and associated helicase (znhel ), exoribonuclease (exon), n -methyltransferase (nmt) and '-o-methyltransferase (omt) domains. the orf a/orf b overlap includes a putative 'slippery' sequence (ggauuuu), allowing orf b expression as a pp ab polyprotein through a − ribosomal frame shift. orf a encodes the s glycoprotein. in cavv, the s protein (p ) has been shown to be generated from the fulllength orf a polyprotein by internal signalase cleavage at the site nahc|strid and is further processed to generate cleavage products s (p ) and s (p ), each of which is a structural component of virions [ ] . a muscle alignment of all available mesonivirus orf a polyprotein sequences (additional file : figure s ) indicated that the signalase cleavage site is relatively variable conforming only to the sequence motif [c/s]|[l/a/s] tridl and that the s -s cleavage site (r|wdssyv) is highly conserved. the s protein, a class i transmembrane glycoprotein with a c-terminal transmembrane domain, features a set of conserved cysteine residues which are likely to form disulphide bridges, and to potential n-glycosylation sites, only of which are conserved across all mesoniviruses. surprisingly, an nterminal signal peptide is strongly predicted for the menov s protein (between amino acids a and s ) but not for those of any of the other mesoniviruses (http:// www.cbs.dtu.dk/services/signalp). there is a relatively high level of amino acid sequence conservation in the s and s proteins ( . % and . % global identify, respectively). however, the n-terminal product of signalase cleavage of the orf a polyprotein displays very low sequence conservation amongst the mesoniviruses ( . % global identity). this product, which is predicted to be positioned on the inside of the er membrane (tmhmm server; www.cbs.dtu.dk/services/tmhmm- . ) has not yet been identified in virions or infected cells. in each of the mesoniviruses, the precise region of the genome encoding the highly variable n-terminal signalase cleavage product of the orf a polyprotein also contains an alternative open reading frame (orf b) which has been shown in cavv to encode the putative nucleoprotein p [ ] . our analysis indicated that the predicted molecular weights of the unmodified mesonivirus n proteins range from . kda (ngev) to . kda (ksav) and all are highly basic (pi > ). they display a moderate level of overall sequence conservation ( . % global identity) due primarily to two highly conserved domains corresponding in ksav to g to a ( . % global sequence identity) and l to f ( . % global sequence identity). the third coding region, commencing immediately downstream of orf a, contains two overlapping long open reading frames (orf a and orf b) each encoding small hydrophobic proteins (figure ) . a clustal x alignment of the mesonivirus orf a proteins and individual structural analyses using signalp and tmhmm and netnglyc (www.expasy.org) indicated that each is a class i transmembrane glycoprotein with a predicted n-termimal signal peptide, an ectodomain containing a conserved set of cysteine residues and a single conserved n-glycosylation site, a transmembrane domain and a c-terminal cytoplasmic domain ( figure a, d) . the cysteine-rich region of the ectodomain displays moderately high sequence conservation including a stretch of totally conserved amino acids adjacent to the transmembrane domain that is unusually rich in large aromatic residues (y, w, f). the cytoplasmic domain is highly variable in sequence, except for the completely conserved motif (h/s)yipllpr. no similar motif has been detected in a search of available eukaryote sequences. the orf b proteins are each predicted to be class ii transmembrane proteins with an n-terminal cytoplasmic domain, a transmembrane domain and a short cterminal ectodomain ( figure b, d) . a single nglycosylation site was predicted in the ectodomain of all the mesoniviruses, except nsev. the orf b proteins display poor overall sequence conservation and have few features that suggest a biological function. the long '-terminal region of all mesoniviruses, except menov, contained a small open reading frame, which was previously designated as orf . a clustal × multiple sequence alignment of the putative proteins indicated that, although they varied in size ( aa to aa), there was a remarkable level of sequence identity, particularly in the n-terminal portion of the protein (additional file : figure s ). however, there are no obvious structural various other short orfs (< nt) were detected in the '-terminal regions of each mesonivirus but none was obviously conserved. alternative orfs of > nt (i.e., amino acids) were also detected within other orfs, including in orf a in the original strain of ndiv ( aa) and the ngewontan ( aa) and houston ( aa) strains, in which the ' region is missing (figure ) . these orfs share a relatively high degree of nucleotide sequence conservation ( %) but a lower level of amino acid sequence conservation ( %) and no obvious structural characteristics indicate the possible function of the encoded proteins. another short orf that displayed some degree of sequence conservation was found in the ' region of orf a for all viruses except menov and kphv (figure ). mesoniviruses have been shown previously to express a '-nested set of polyadenylated sub-genomic mrnas produced by copy-choice mediated leader-body fusion at sites located immediately upstream of alternative transcription regulatory sequence (trs) elements which occur in the '-utr and in the regions preceding orf a/ orf b and orf a/orf b [ ] . each of the previously identified leader-body fusion sites and trs elements [auxxuacuacuacua and agax(x)acucuccca] were completely conserved across all new mesoniviruses examined in this study. in nidoviruses, translation of orf b characteristically occurs through a ribosomal frame-shift at a 'slippery' sequence in the orf a/orf b overlap region to allow read-through synthesis of a polyprotein (pp ab). this is usually facilitated by an rna pseudoknot in the sequence immediately downstream of the frame-shift site. however, a previous analysis of the ndiv sequence failed to identify a predicted pseudoknot structure, suggesting that a stem-loop structure (predicted using pknotsrg; http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/) in the same region of the genome may facilitate the frame-shift [ ] . however, our analysis using pknotsrg of the new mesonivirus sequences and those of cavv, hanav, ndiv, nsev and menov indicated that, although the slippery sequence (ggauuuu) is completely conserved, the stem-loop structure predicted for ndiv is not predicted as the minimum free energy structure for any of the other viruses. indeed, an alignment of the corresponding region of the genome of all mesoniviruses (additional file : figure s ) indicated that there was poor conservation of this sequence and few compensatory mutations that would preserve the stem-loop structure ( figure a ). as functional pseudoknot structures are usually conserved, an alignment of all mesonivirus sequences extending nt from the start of the 'slippery sequence' was analyzed using the ipknot server (http:// rna.naist.jp/ipknot) which predicts the consensus secondary structure from a multiple sequence alignment (by using integer programming to select the maximum expected accuracy (mea) structure) [ ] . as shown in figure b , the ipknot algorithm predicted a conserved pseudoknot structure, which conformed to all nucleotide substitutions in the available mesoniviruses. analysis of the region of the orf a/orf b overlap indicated that it has a similar format to that of the orf a/orf b overlap region, featuring a long overlap region and potential slippery sequence (cacuuuu) that could result in read-through by a − ribosomal frameshift. structural analyses using signalp and tmhmm and netnglyc (www.expasy.org) indicated that a frame-shift at this site would result in a double-membrane-spanning glycoprotein (p ab) of approximately - kda (depending on whether one or two n-glysosylation sites are occupied) with both the n-terminal and c-terminal domains located in the extracellular lumen (figure c, d) . a previous analysis of cavv proteins by mass spectrometry identified peptide sequences from proteins migrating in gels at , and kda that corresponded to the a protein (designated the m protein) and these were considered to be variously glycosylated forms; no protein corresponding to the orf b gene product was detected [ ] . as for the orf a/orf b region, analysis of the genome region downstream of the putative ribosomal frame-shift element (rfs) element using pknotsrg identified various potential stem-loops but no commonly predicted minimum free energy structure. analysis of a multiple sequence alignment of the region using the ipknot server also failed to predict a convincing pseudoknot structure. in this study we have identified mesonivirus isolates and characterized their genome organization and phylogenetic relationships. based on species demarcation criteria employed previously for mesoniviruses [ ] , five of these new isolates would be assigned to the same species as ndiv and cavv (alphamesonivirus- ), other previously described mesoniviruses -hanav menov, and nsev -would be assigned to three new species (alphamesonivirus- , alphamesonivirus- and alphamesonivirus- , respectively), and eight of the new isolates would represent three new species (alphamesonivirus- , alpha mesonivirus- and alphamesonivirus- ) [ ] . however, we consider this basis for species demarcation, which employs only a genetic standard of pairwise sequence divergence to assign viruses to a species, should be re-evaluated following further assessment of the ecology of these viruses and their potential for genetic recombination which may provide a more informed analysis of suitable species demarcation criteria. the isolates of viruses assigned to the species alphamesonivirus- illustrate the wide geographic distribution and mosquito host range of some mesoniviruses. the original four isolates of ndiv were made in northern and central vietnam from culex vishnui and cx. tritaeniorhynchus mosquitoes collected indoors during a surveillance program for japanese encephalitis virus (jev) [ ] . our ndiv isolate from java, indonesia (ngewontan strain) was also obtained from a pool of cx. vishnui mosquitoes in . the four ndiv isolates from houston, texas (houston strain) were made from cx. quinquefasciatus and aedes albopictus collected outdoors within the houston metropolitan area during west nile virus (wnv) surveillance in and , respectively. interestingly, all of these isolates were from mosquitoes captured in or near human dwellings and from species that feed on humans. the close similarity of isolates from houston and vietnam may suggest a recent translocation, possibly during the vietnam conflict when houston hosted a major air base for embarkation/disembarkation. isolates of kampaeng phet, botang baru and karang sari viruses, which may be considered three new mesonivirus species, have a geographic distribution extending at least from central thailand to kalimantan and java in indonesia, and have been isolated from at least two species of culex mosquitoes. another consideration is the potential effect of mesonivirus infection on the susceptibility and vector competence of mosquitoes for viral pathogens of vertebrates. for example, both cx. vishnui and cx. tritaeniorhynchus are important vectors of jev in asia, and cx. quinquefasciatus is the major vector of wnv in houston. recent experimental studies with ae. aegypti mosquitoes infected with certain strains of the wolbachia indicate that the presence of the symbiont bacterium interferes with dengue virus replication and decreases . stem-loop structure predicted previously for ndiv [ ] using pseudoknotsrg software, illustrating nucleotides that are substituted in various viruses (red circles). nucleotide substitutions (which are primarily non-compensatory) are shown in the boxes. the corresponding sequence alignment is shown in additional file : figure s . (b). an alternative conserved pseudoknot structure predicted from the multiple sequence alignment using ipknot software. the putative 'slippery' sequence (ggauuuu) at the ribosomal frame-shift site is shaded in grey. vector competence, possibly by upregulating or priming the mosquito's innate immune system [ , ] . similar results have been reported for wolbachia-infected ae. aegypti and chikungunya virus [ ] and with wolbachia-infected cx. quinquefaciatus and wnv [ ] . if a bacterial endosymboint can alter a mosquito's vector competence for arboviruses, it seems plausible that a viral symbiont could have a similar effect [ ] . this is an important area for future investigation. due to the continuous efforts of the virus discovery program of world reference center for emerging viruses and arboviruses (wrceva), we have continued to isolate mesoniviruses from various insect vectors collected from widespread geographic locations (e.g., nepal, colombia and south florida) suggesting that these viruses are more common than previously thought. zirkel et al. [ ] suggested that these viruses may have their origins in pristine rainforests and emergence may have been facilitated through anthropogenic-induced modifications (e.g., altered land use, deforestation). the detailed analysis and comparison of mesonivirus genome architecture conducted here has revealed some unexpected characteristics. firstly, the presence of block insertions of up to nt in the ' terminal quadrant of orf a of several mesoniviruses has not been reported previously. the function of this region is presently unknown in mesoniviruses and other nidoviruses and so the structural and functional consequences of these insertions, which contain various imperfect repeats is unclear. although sharing similar genome architecture, nidoviruses vary greatly in genome size. a previous analysis of the evolution of nidovirus genomes concluded that genome expansion has occurred in a wave-like fashion in which the three major coding regions (orf b, orf a and the 'orfs) expanded consecutively in a hierarchy that reflects the roles of their encoded proteins in the virus replication cycle [ ] . this implies that nidoviruses have an inherent capacity for genome expansion, most likely associated with the transitional retention of sequences that serve as a resource for the evolution of new functions. the block insertions detected in orf a appear to be functionally redundant and their potential role in such evolutionary processes is presently unclear. the comparative analysis also revealed that the stemloop structure which had previously been identified at the rfs site of ndiv is not conserved in the other mesoniviruses and so may not be responsible for activating the − ribosomal frame shift. secondary structure predictions using ipknot on the aligned sequences downstream of the conserved 'slippery' sequence site revealed a conserved pseudoknot structure that conformed to all nucleotide substitutions. however, the predicted structure featured only four relatively short regions of complementarity and no estimations of minimum free energy for the represented structures are available through this algorithm. a possible 'slippery' sequence (cacuuuu) was also detected in the orf a/ orf b overlap region but no conserved stem-loop or pseudoknot structure was predicted by ipknot in the downstream sequence. it is, therefore, unclear whether orf b is expressed by internal initiation or as a read-through extension of orf a, which would generate a double-membrane spanning protein. functional analysis of each of the regions corresponding to the rfs in orf a/orf b and the putative rfs in orf a/orf b would help resolve the mechanisms of mesonivirus gene expression. in conclusion, we have identified and characterized several new mesoniviruses from mosquitoes of human medical importance sampled over time from widespread geographic regions. several important questions related to their transmission, maintenance in insect hosts in nature, their potential impact of infection on the insect's behavior, fertility, fecundity and survival, their evolution, their mechanisms of gene expression and their potential to be developed as biological control agents, warrant further investigation. all viruses used in this study were obtained from the wrceva at the university of texas medical branch. some were isolated by the authors (rbt and hg), during arbovirus field studies; the remainder were isolated by other investigators and sent to the wrceva for identification and further characterization. all isolations were originally made in mosquito cell cultures (c / or ap- ). the proposed names, original sources and geographic origins and genbank accession numbers of the sequences obtained for the viruses included in our study are listed below and in table . jkt- was isolated from a pool of culex vishnui mosquitoes collected on / / at karang sari, cilacap (central java) indonesia. the initial isolation was made at the naval medical research unit # (namru- ) in jakarta. jkt- was isolated at namru- from a pool of culex vishnui collected at bontag baru, east kalimantan, indonesia in february . strains jkt- , jkt- and jkt- were also isolated at namru- appear to be almost identical to isolate jkt- in the phylogenetic tree. since jkt- was the first virus in this group to be isolated, it should be the prototype. jkt- was isolated at namru- from a pool of tvp and tvp -these two viruses were also isolated at utmb from pools of aedes albopictus mosquitoes collected in june as part of the harris county arbovirus surveillance program in houston, texas. before sequencing, all virus stocks were grown in cultures of the c / clone of ae. albopictus cells [ ] , obtained from the american type culture collection (atcc), manassas, va. infection was characterized by detachment of cells and cell lysis. for ultrastructural analysis in ultrathin sections infected cells were fixed for at least hr in a mixture of . % formaldehyde prepared from paraformaldehyde powder, and . % glutaraldehyde in . m cacodylate buffer ph . to which . % picric acid and . % cacl were added. the monolayers were washed in . m cacodylate buffer, cells were scraped off and processed further as a pellet. the pellets were post-fixed in % oso in . m cacodylate buffer ph . for h, washed with distilled water and en bloc stained with % aqueous uranyl acetate for min at °c. the pellets were dehydrated in ethanol, processed through propylene oxide and embedded in poly/bed (polysciences, warrington, pa). ultrathin sections were cut on leica em uc ultramicrotome (leica microsystems, buffalo grove, il), stained with lead citrate and examined in a philips transmission electron microscope at kv. viral rna ( . - . μg) was fragmented by incubation at °c for min in . ul of fragmentation buffer (illumina ). first and second strand synthesis, adapter ligation and amplification of the library were performed using the illumina truseq rna samplec preparation kit under conditions prescribed by the manufacturer (illumina). samples were tracked using the "index tags" incorporated into the adapters as defined by the manufacturer. mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses examining landscape factors influencing relative distribution of mosquito genera and frequency of virus infection discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes an insect nidovirus emerging from a primary tropical rainforest identification and characterization of genetically divergent members of the newly established family mesoniviridae molecular biology and pathogenesis of roniviruses a new nidovirus (namdinh virus ndiv): its ultrastructural characterization in the c / mosquito cell line a new species of mesonivirus from the northern territory, australia supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy rtips: fast and accurate tools for rna d structure prediction using integer programming a wolbachia symbiont in aedes aegypti limits infection with dengue, chikungunya, and plasmodium the relative importance of innate immune priming in wolbachia-mediated dengue interference the native wolbachia endosymbionts of drosophila melanogaster and culex quinquefasciatus increase host resistance to west nile virus infection negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution the footprint of genome architecture in the largest genome expansion in rna viruses isolation of a singh's aedes albopictus cell clone sensitive to dengue and chikungunya viruses smart : recent updates to the protein domain annotation resource smart, a simple modular architecture research tool: identification of signaling domains muscle: multiple sequence alignment with high accuracy and high throughput new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing sse: a nucleotide and amino acid sequence analysis platform mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range additional file : figure s . a clustal x multiple sequence alignment of region immediately downstream of the orf a/orf b rfs that has been predicted previously to adopt a stem-loop structure in ndiv. the alignment illustrates sequence variations in nucleotides predicted in ndiv to be involved in base pairs that sustain the structure. cluster formation of the library dna templates was performed using the truseq pe cluster kit v (illumina) and the illumina cbot workstation using conditions recommended by the manufacturer. paired end base sequencing by synthesis was performed using truseq sbs kit v (illumina) on an illumina hiseq using protocols defined by the manufacturer. cluster density per lane was - k/mm and post filter reads ranged from - million per lane. base call conversion to sequence reads wasperformed using casava- . . . virus sequences were edited and assembled using the seqman and nextgen modules of the dnastar lasergene program (bioinformatics pioneer dnastar, inc., madison, wi). in certain cases, prefiltering of reads to remove host sequence enhanced the assembly process. assembly was carried out using a fasta file of aedes albopictus sequences to remove host dna from the assembly thus reducing the number of contigs present. the compiled sequences had their relationship to other viruses determined by a blastx search. the open reading frames were determined using enzyme x (nucleobytes, inc., aalsmeer). relationships to the other two insect-specific nidoviruses were determined using macvector (cary, nc) using their dna identity matrix software. the presence of conserved protein domains was determined using the smart webserver [ , ] . a region corresponding to the full-length product encoded by orf a (unprocessed s proteins) was used to determine the relationships within the family mesoniviridae along with a concatenated region of the highly conserved domains within orf ab ( cl pro , rdrp and znhel ). orf ab alignments were determined using the muscle algorithm [ ] as amino acids before being toggled back to the nucleotides while maintaining the alignment. concatenated orf ab alignments were performed at the protein level. a maximum likelihood (ml) tree for orf a was constructed in mega . using the jones-taylor-thornton substitution model of nucleotide substitution, uniform substitution rates among sites and the nearest neighbor-interchange heuristic method and a very strong branch swap filter. an optimal ml tree was then estimated using the appropriate model and a heuristic search with tree-bisection-reconstruction branch swapping and replicates, estimating variable parameters from the data, where necessary. a maximum likelihood phylogeny of the conserved domains of orf ab was constructed using phyml . [ ] , the wag + gamma model of amino acid substitution, and the nni + spr method of branch swapping. one thousand bootstrap replicates were calculated for each dataset under the same models and expressed as a percentage. calculation of the ped between the highly conserved protein domains of orf ab ( cl pro , rdrp and znhel ) of the thirteen new isolates described in this study and those of five previously described mesoniviruses (cavv, hanav, ndiv, nsev and menov) was performed using the ml method in the program treepuzzle [ ] and the wag model of amino acid substitution. sliding window analysis was used to calculate the mean amino acid divergences within and between the seven mesonivirus species described in this study. percent divergences were calculated for each of the three largest orfs in the genome (orf ab, orf a, orf b) using the program sse [ ] . additional file : figure s . sliding window analysis of the pairwise amino acid distances within and between the seven putatively designated mesonivirus species for orf ab (replicase proteins), orf a (s) and orf b (n).additional file : figure s . a clustal x multiple sequence alignment of mesonivirus pp ab polyproteins illustrating region containing the block insertions (yellow shading) and various imperfectly repeated sequences that occur at the boundary and within the blocks of inserted sequence.additional file : figure s . a clustal x multiple sequence alignment of the polypeptides encoded in orf a (s proteins) of the mesoniviruses. a predicted signal peptide in menov and predicted transmembrane domains in all mesoniviruses are shaded in aqua, predicted n-glycosylation sites are shaded in green, cysteine residues are shaded in yellow and the sites of proteolytic cleavage to generate glycoproteins s and s and the unidentified n-terminal fragment are shaded in purple.additional file : figure s . a clustal x multiple alignment of the sequences of putative polypeptides encoded on orf which occurs in the '-terminal regions of all mesoniviruses except menov. to emphasize the alignment, the ksav orf protein has been shown to commence at the next available methionine residue located amino acids downstream of the predicted initiation codon. the authors do hereby declare that they have no competing interests in this scientific work. key: cord- -pibq iwh authors: jumat, muhammad raihan; nguyen huong, tra; wong, puisan; loo, liat hui; tan, boon huan; fenwick, fiona; toms, geoffrey l; sugrue, richard j title: imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of f-actin and the raft-lipid microdomains in virus morphogenesis date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: pibq iwh backgound: due to difficulties of culturing human metapneumovirus (hmpv) much of the current understanding of hmpv replication can be inferred from other closely related viruses. the slow rates of virus replication prevent many biochemical analyses of hmpv particles. in this study imaging was used to examine the process of hmpv morphogenesis in individually infected llc-mk cells, and to better characterise the sites of hmpv assembly. this strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the hmpv particles and the sites of hmpv morphogenesis. methods: hmpv-infected llc-mk cells were stained with antibodies that recognised the hmpv fusion protein (f protein), attachment protein (g protein) and matrix protein (m protein), and fluorescent probes that detect gm within lipid-raft membranes (ctx-b-af ) and f-actin (phalloidin-fitc). the stained cells were examined by confocal microscopy, which allowed imaging of f-actin, gm and virus particles in hmpv-infected cells. cells co-expressing recombinant hmpv g and f proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant g and f proteins and phalloidin-fitc and ctx-b-af , and the distribution of the g and f proteins, gm and f-actin determined. results: hmpv-infected cells stained with anti-f, anti-g or anti-m revealed a filamentous staining pattern, indicating that the hmpv particles have a filamentous morphology. staining of hmpv-infected cells with anti-g and either phalloidin-fitc or ctx-b-af exhibited extensive co-localisation of these cellular probes within the hmpv filaments. this suggested that lipid-raft membrane domains and f-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the hmpv filaments. furthermore, the filamentous virus-like particles that form in cells expressing the g protein formed in cellular structures containing gm and f-actin, suggesting the g protein contains intrinsic targeting signals to the sites of virus assembly. conclusions: these data suggest that hmpv matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of f-actin. the similarity between hmpv morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of f-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the pneumovirinae. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. human metapneumovirus (hmpv) is a new member of the paramyxoviridae that was first identified in children with respiratory diseases in netherlands [ ] . the clinical symptoms that are caused by hmpv infections in children are similar to those observed with respiratory syncytial virus (rsv) infection, ranging from upper respiratory tract infection to bronchiolitis and pneumonia. hmpv has become recognised as a major cause of lower respiratory infection in children [ , ] . the mature hmpv particle is surrounded by a lipid envelope in which the virus fusion (f) and attachment (g) proteins are inserted. the f protein mediates fusion of the virus and host cell membranes during virus entry [ ] , while a primary role for the g protein in virus attachment to susceptible cells has been demonstrated [ ] . the virus envelope surrounds a protein layer formed by the matrix (m) protein, and a ribonucleoprotein (rnp) complex that is formed by the viral genomic rna (vrna), the nucleocapsid (n) protein, the phosphoprotein (p protein), the m - protein and the large (l) protein [ ] . based on genetic analysis of hmpv genome sequences two major hmpv genotypes, called hmpv a and b, have been identified [ ] [ ] [ ] . much of the current understanding of the biology of the hmpv can be inferred from other closely related viruses e.g. rsv and avian pneumovirus [ , ] . primary isolation of hmpv has been achieved in several different cell lines [ , , ] , and some tissue culture adapted isolates have been described [ ] . however, their cultivation can require up to days incubation before cytopathic effects are visualised [ ] . this low level of virus replication in standard cell culture, particularly low-passaged clinical isolates, and the subsequent recovery of low levels of infectious hmpv, have hampered functional biochemical studies on the virus. these studies usually require higher levels of biological material that can be achieved following a single cycle of hmpv replication. visualising the distribution of individual virus structural protein is a prerequisite for understanding the process of hmpv maturation, and in situ imaging of virus-infected cells stained using virus protein specific antibodies is in general the most direct and unambiguous method to do this. therefore, in this current study we have circumvented the problems associated with low virus replication rates by using imaging to examine hmpv morphogenesis. this has allowed us to visualize the morphogenesis of a low passaged hmpv clinical isolate in mammalian tissue culture, and to suggest a role for lipid-raft microdomains and f-actin in the process of hmpv maturation. the hmpv a strain ncl - / used in this study was isolated from nasal secretions of children with respiratory infection, and the virus was cultured as described previously [ ] . hmpv isolation and propagation in the llc-mk cell line has been described by several groups [ , , ] , and this cell line was used throughout our study. the distribution of several major virus structural proteins was characterised using antibodies against the f, g, and m proteins to stain hmpv-infected cells. the preparation of the hmpv f (mab and mab ) and g (mabat ) protein antibodies has been described previously [ ] , and the antibody against the hmpv m protein (anti-m) was prepared using bacterially expressed recombinant hmpv m protein. semi-confluent cell monolayers were infected with hmpv using a multiplicity of infection (moi) of . , and at , and days-post infection (dpi) the monolayers were fixed and stained using anti-m (additional file : figure s ). by dpi we were able to detect individually infected cells which exhibited low levels of anti-m staining. however, by dpi infected cells showed increased levels of fluorescence staining, and the appearance of brightly stained infected cell clusters within the monolayer was noted by dpi. interestingly, the low rates of infection of the hmpv ncl - / isolate in the llc-mk cell monolayer suggested that although the virus can infect these cells it is not extensively tissue culture-adapted. at dpi approximately . x infectious virus particles per ml was detected in the tissue culture supernatant of hmpv-infected cells. however, the infected cell clusters were the most prominent staining pattern detected on the cell monolayers, and the increase in cluster size was time-dependant. this provided evidence that hmpv transmission occurred by localised transmission within the llc-mk cell monolayer, in a manner similar to that described in rsv-infected cell monolayers [ ] . since dpi allowed clear visualisation of infected cells by antibody staining without extensive cpe, all our subsequent analyses were performed at this time of infection unless otherwise stated. to demonstrate the specificity of the antibodies used in this study, detergent extracts were prepared from mock-infected and virus-infected cells, and the virus proteins examined by immunoblotting and immunoprecipitation ( figure ). mock and hmpvinfected cells were detergent exacted and the presence of the g protein detected by immunoblotting using mabat . this revealed the presence of the kda monomeric (g) and a kda g protein species (g*) ( figure a ). it is currently unclear if g* is a differentially glycosylated form of the g protein or if it is an oligomeric form of the g protein that shows increased resistance to heat denaturation. immunoblotting of detergent extracts prepared from mock and hmpvinfected cells with anti-m revealed the presence of a kda protein in the infected cells, consistent with the presence of the m protein ( figure b ). in addition the cells were surface-biotinylated, detergent-extracted, and the surface-labelled g and f proteins isolated from the detergent extract by immunoprecipitation using mabat and mab respectively as described previously [ ] . immunoprecipitation with mabat revealed the presence of g and low levels of g* ( figure c ), while immunoprecipitation with mab revealed the presence of the kda f subunit similar to that described previously [ ] . immunoprecipitation with mab also revealed the presence of an additional and a kda surface biotinylated species that appeared to co-precipitate with the f subunit. the f precursor and f protein subunit were also detected in [s ] methionine-labelled hmpv-infected cells immunoprecipitated with mab ( figure d ). hmpv-infected cells stained using anti-m, anti-f or anti-g were examined using confocal microscopy at an optical plane that allowed imaging of the cell surface. staining with each of the three different antibodies revealed a similar filamentous staining pattern in each case ( figure a ). this indicated that hmpv particles can form filamentous structures in a manner similar to that described for the mature rsv particles on infected cells [ ] [ ] [ ] . examination of anti-g stained cells in crosssection further confirmed the presence of these filamentous structures on the surface of infected cells ( figure b ). in a parallel analysis, infected cells were stained using anti-g and evans blue (additional file : figure s b ), the latter being a non-specific counter stain that is used . at dpi mock-infected (m) and hmpv-infected (i) cells were surfacebiotinylated using sulpho-nhs-lc-lc-biotin as described previously ( [ ] ). the surface-labelled detergent extract was immunoprecipitated using (c) mabat (anti-g) or mab (anti-f). the biotin-labelled proteins were immunoblotted and detected using strepavidin-hrp. surface-biotinylated protein species corresponding in size to g, g*, and the f subunit (f ) are indicated. (d) mock and hmpv-infected cells at dpi were labelled for hr using μci/ml l-[ s]-methionine (easytag, perkinelmer) in methionine free-dmem (invitrogen) and the detergent extracts immunoprecipitated using mab as described previously [ ] . the f and f subunit are indicated. in plate c and d additional protein products that are immunoprecipitated with anti-f are indicated (*). to stain the cell monolayer (i.e. both non-infected and infected). it was noted that these filamentous virus structures appeared to spread from the infected cells to the surrounding non-infected cells. although the significance of this vis-a-vis hmpv transmission is unclear, in the closely related rsv, virus filaments have been shown to play a direct role in virus cell-to-cell transmission [ ] . interestingly, closer inspection of the anti-m stained hmpv-infected cells revealed that in addition to a filamentous staining pattern ( figure a) , the presence of a spherical staining pattern at the ends of some of these filaments ( figure b ). these varied in size, but we estimated they had an approx. diameter of between and nm, and were located most commonly around the periphery of the infected cells ( figure b (ii), highlighted by white arrow heads). infected cells stained using anti-f or anti-g did not show this alternative prominent staining pattern. a comparison of the surface topology of mock-infected and hmpv-infected cells was performed using scanning electron microscopy (sem) (additional file : figure s ). this confirmed the presence of hmpv-induced filamentous structures on the surface of infected cells; and was consistent with filamentous staining pattern observed using light microscopy. interestingly, the sem analysis also showed the presence of spherical structures associated with the virus filaments, and which may represent the locations of the m protein special structures detected by confocal microscopy. the significance of the spherical m protein staining pattern is unclear, but does suggest that in addition to the virus particle associated m protein, a second population of the m protein that does not show a similar staining to that observed using either anti-g or anti-f staining. this alternative m protein staining pattern may be related to the cell-free form of the m protein that has been described in hmpv-infected cells [ ] . in addition, our previous studies employing recombinant expression to generate hmpv virus like particles (vlps) [ ] identified a population of the hmpv m protein that was secreted within membrane vesicles but which did not co-purify with the vlps [ ] . the role of lipid-raft membranes and the cortical factin network during rsv morphogenesis has been described [ ] [ ] [ ] [ ] [ ] . we therefore examined if f-actin and lipid-raft microdomains were also associated with hmpv filaments during virus assembly. phalloidin-fitc binds to f-actin and cholera toxin-b subunit (ctx-b-af ) binds to the raft-lipid gm , enabling the detection of f-actin and lipid-raft membranes respectively. these fluorescence probes were used to examine the distribution of f-actin and lipid-raft membranes with respect to the hmpv filaments in hmpvinfected cells. cells were infected with hmpv using a moi of . and at dpi the cells were stained using phalloidin-fitc and anti-g. the stained cells were imaged using confocal microscopy at optical planes which allowed the cell periphery ( figure a ) and cell top ( figure b and c) to be visualized. the stained monolayer allowed the detection of both infected and non-infected cells, and we noted a similar prominent filamentous staining pattern for both anti-g and phalloidin-fitc in hmpv-infected cells. the lsm software was used to measure the degree of co-localisation within this filamentous staining pattern. a mander's coefficient of . ± . and pearson's coefficients of . ± . indicated high levels of colocalisation in these filamentous structures. our imaging analysis suggested that f-actin was present within the hmpv filaments in a similar manner to that described for rsv [ , , ] . due to the low replication rates of hmpv not all cells in the monolayer were infected at dpi. this enabled us to distinguish the infected cells (anti-g stained positive) from the remaining non-infected cells (anti-g stained negative) within the same cell monolayer ( figure a) . it was noted that this prominent filamentous f-actin staining pattern was not detected in non-infected cells at a similar optical plane, suggesting that hmpv infection may alter the structure of the cortical actin network in a similar manner to that described in rsv-infected cells [ , , ] . we also observed these co-stained filamentous structures spreading to the non-infected cells, suggesting that f-actin may play a role in virus transmission in the monolayer, in a similar manner to that proposed for rsv [ , , ] . hmpv-infected cells were stained with anti-g and ctx-b-af , and examined using confocal microscopy at an optical plane that allowed the virus filaments to be visualized ( figure d ). we observed a similar filamentous staining pattern for both ctx-b-af and anti-g, and a high level of co-localisation within this filamentous staining pattern as indicated by both mander's coefficient and pearson's coefficients of . ± . and . ± . respectively. our data also suggested that hmpv assembly occurs within membrane microdomains that are enriched in lipid-rafts, and that these lipids are incorporated into the virus envelope. the hmpv g protein preferentially localises to cellular microdomains enriched in f-actin and gm using recombinant expression we have recently demonstrated that co-expression of the hmpv g and f proteins in llc-mk cells leads to the formation of viruslike particles (vlps) [ ] . these vlps form filamentous structures on the surface of transfected cells that are smaller but similar in appearance to the filamentous hmpv particles. we noted that expression of the g protein was sufficient to form vlps, and vlps formed in cells co-expressing the g and f proteins contained a protein complex involving the hmpv f and g proteins. we interpreted these vlp structures as indicating sites of hmpv assembly to which the g protein and f proteins were trafficked, rather than representing the formation of mature virus particles. this further suggested that the g and f proteins contain trafficking signals that allow their targeting to these sites of virus assembly. by extrapolation we rationalised that these signals would also play a role in their incorporation into hmpv particles when they form in hmpv-infected cells. on this basis we concluded that the recombinant g protein could be used as a marker with which to identify the sites of virus assembly. we therefore used this recombinant expression system to examine the relative distribution of the g protein, gm and f-actin in cells expressing recombinant g protein. cells were transfected with pcaggs/g-flag and at hrs post-transfection the cells were co-stained with anti-flag (to detect the g protein) and phalloidin-fitc ( figure a-d) . similarly, cells were co-transfected with pcaggs/g-flag and pcaggs/f-cmyc and stained with anti-cmyc (to detect the f protein) and phalloidin-fitc ( figure e and f) . in both cases a prominent filamentous g and f protein staining pattern was observed as described previously [ ] , and in both cases we noted co-staining of the g and f protein filaments with phalloidin-fitc. similarly cells were transfected with pcaggs/g-flag and at hrs post-transfection the cells were co-stained with anti-flag and ctx-b-af ( figure a and b) . a prominent filamentous g protein staining pattern was observed, and we noted extensive co-staining of the g protein filaments with ctx-b-af . we confirmed the interaction between the f and g proteins by co-immunoprecipitation assay (figure ) . the cells were transfected singly with pcaggs/g-flag or pcaggs/f-cmyc, or alternatively co-transfected with pcaggs/g-flag and pcaggs/f-cmyc, and after hr post-tranfection the cells were surface-biotinylated ( figure a and b) . cells expressing only the f protein or g protein immunoprecipitated with either anti-flag or anti-cmyc respectively showed biotinylated protein species corresponding in size to the g and f proteins respectively. in lysates prepared using cells co-expressing the pcaggs/ g-flag and pcaggs/f-cmyc and immunoprecipitated with anti-flag a protein species corresponding in size to the g protein was observed. in contrast immunoprecipitation with anti-cmyc also showed protein species corresponding in size to the g protein. this was consistent with an interaction between the f and g proteins on the surface of co-transfected cells. as observed previously [ ] , we also noted that the g protein expressed in co-transfected cells exhibited a slightly larger apparent mass compared to that in cells expressing only the g protein. we originally interpreted this observation as evidence that co-expression of the f protein influenced the processing of the g protein, providing additional evidence of an interaction. we failed to detect the presence of biotinylated proteins in cells transfected with the parent expression vector and immunoprecipitated with either anti-flag or anti-cmyc ( figure c ). co-precipitation of the g protein following immunoprecipitation with anti-cmyc and f protein following immunoprecipitation with anti-flag was confirmed by immunoblotting with anti-flag and anti-cmyc respectively ( figure d ). these observations suggested the formation of a single protein complex between the f and g proteins in the co-transfected cells, confirming our earlier observations [ ] . the imaging analysis suggested that the protein complex formed by the f and g proteins is located within f-actin stabilised cellular structures on the surface of cotransfected cells. we have recently demonstrated that the hmpv vlps could be isolated by ultracentrifugation using a %(w/v)sucrose, %(w/v)sucrose, and %(w/v) sucrose discontinuous gradient ( ) . in this earlier study we had used this discontinuous sucrose gradient centrifugation methodology as an assay with which to monitor vlp formation and identify virus determinants of vlp formation ( ) . cells were co-transfected with pcaggs/ g-flag and pcaggs/f-cmyc and after hr posttransfection the cells were harvested and vlps prepared as described in methods. the gradient was fractionated and the interfaces between the different sucrose concentrations examined by immunoblotting using anti-cmyc and anti-flag to detect the presence of the f-myc and g-flag respectively. similarly, we examined the different fractions by immunobloting using anti-actin to determine the presence of actin. immunoblotting with anti-flag ( figure a ), and anti-cmyc ( figure b ) revealed the presence of the hmpv g and f proteins in the / (w/v) sucrose interface as we have demonstrated previously ( ) . in addition, immunoblotting of these fractions with antiactin ( figure c ) showed the presence of actin in the same fraction as the hmpv glycoproteins i.e. at the / (w/v) sucrose interface. this provides additional biochemical evidence that the vlps are formed within the f-actin structures identified in the imaging analysis of the cotransfected cells. these data indicated that the g protein is able to transport to regions of the cell membrane that are enriched in f-actin and gm independently of other virus proteins. as suggested in our previous study, the filamentous staining pattern exhibited by the hmpv g protein indicates the presence of vlps rather than the filamentous particles that are produced in hmpv-infected cells. we propose that the vlps allow the identification of the sites of hmpv particle assembly in llc-mk cells. this further suggested that the hmpv g protein contains the necessary targeting signals that can traffic the protein into the sites of hmpv particle assembly, locations where lipidraft microdomains and f-actin are enriched. our observations indicate that the hmpv virus matures with a filamentous morphology in a manner similar to that described for rsv. several studies have suggested a role for f-actin in rsv particle formation [ , ] . factin is also present at the site of hmpv assembly, and the correlation between hmpv assembly and the presence of f-actin suggests that f-actin may also play a role in the formation of hmpv filaments. lipid-raft membranes exist as a liquid crystalline phase, leading to formation of stabilized lipid structures within the bulk lipid membrane [ ] . the incorporation of cholesterol-dependent raft structures into the influenza virus envelope correlates with a reduction in the 'fluidity' of the viral envelope [ ] . the presence of raftlipids in the envelope of hmpv filaments suggested the presence of similar highly ordered lipid microdomains (lipid-rafts) within the viral envelope. this further suggests that these lipids may impart important structural and functional properties to the hmpv envelope. the low virus yields and slow replication rates of hmpv in tissue culture are major impediments to understanding hmpv morphogenesis using standard biochemical methodology. however, in this study we have used imaging to examine the distribution of virus structural proteins and specific cellular markers during hmpv maturation in individual virus-infected cells. this experimental approach has provided the first direct evidence to suggest a role for lipid-raft membranes and f-actin in the process of hmpv morphogenesis. interestingly, the similarity between hmpv and the closely related rsv suggests that the formation of virus filaments may be a common feature of virus morphogenesis for other viruses within the family pneumovirinae. our analysis also highlights the utility of in situ imaging using specific virus and cellular markers to examine the morphogenesis of viruses that are not easy to propagate in standard tissue culture. this alternative strategy should become increasingly accessible as the number of commercially available immunological reagents and cellular probes increases. the hmpv a strain ncl - / was described previously [ ] . the hmpv was used to infect the llc-mk cell line in dmem (bsa, . μg/ml tpkc-trypsin) at °c. the llc-mk cell line was maintained in dmem with % fcs at °c. the antibodies mab (anti-f) and at (anti-g) have been described previously [ ] . the tagged hmpv proteins were detected with rabbit anti-flag antibodies (sigma-aldrich, usa), mouse anti-cmyc antibodies (cell signaling technology, usa). the anti-m was prepared using recombinant expressed m protein. briefly, the hmpv m gene was cloned into prsetb and expressed in mm iptg-induced e.coli as his-tagged proteins. after hr induction the bacterial cells were lysed and the recombinant hmpv proteins recovered from the insoluble lysed bacterial pellet using m guhcl ( mm edta, mm nacl, mm tris-cl ph ). the proteins were bound to nickel agarose using the manufacture's protocol (qiagen), and the recombinant proteins were eluted using m urea in mm imidazole, mm edta, mm nacl, mm tris-cl ph , and diluted into refolding buffer ( mm edta, mm pmsf, mm nacl, mm tris-cl ph ). the resulting eluted protein solution was concentrated and used to immunise balb/c mice and monoclonal antibodies prepared using standard procedures. the g and f gene were amplified from the hmpv a positive-nasopharyngeal washings (sin -ntu ) and inserted into the vector pcaggs [ ] to generate pcaggs/g-flag and pcaggs/f-cmyc respectively as described previously [ ] . cells were transfected using lipofectamine reagent (invitrogen, usa) following the manufacturer's instructions. the proteins were separated by sds-page, transferred onto pvdf membranes (immobilon-p, milipore, usa) as described previously [ ] . protein bands were visualised using the ecl system (ge healthcare, usa). molecular masses were estimated using kaleidoscope markers (biorad, usa). cell extracts were prepared at °c using rip buffer ( % np- , . %sds, mm nacl, mm edta, mm pmsf, mm lysine, mm tris-hcl, ph . ) and clarified by centrifugation ( , g, min °c) and immunoprecipitated as described previously [ ] using appropriate antibodies. the immunoprecipitation assays were separated using sds-page. cells were surface-labelled using ez-link sulfo-nhs-lc-lc-biotin (pierce biotechnology, usa) as described previously [ ] . briefly, cell monolayers were incubated in . mg/ml solution of ez-link sulfo-nhs-lc-lc-biotin (pierce biotechnology, usa) in pbs ph for hr at room temperature. the lysates were immunoprecipitated using the appropriate antibody. cells were fixed with % pfa or methanol:acetone ( : ) for min at °c and the cells were labelled using the appropriate primary and secondary antibody (conjugated to either fitc or alexa fluor ). staining with phalloidin-fitc (sigma) and cholera toxin-b subunit conjugated to af or af (invitrogen) was also performed as described previously [ ] . the cells were visualized using either a nikon eclipse i fluorescence microscope (nikon eclipse te -u) or a zeiss axioplan confocal microscope using appropriate machine settings. this was performed as described previously ( ) . briefly, cell suspension was subjected to freeze-thaw, the cell suspension was clarified ( , g for min) and loaded onto a sucrose cushion ( %w/v sucrose in ten buffer), and centrifuged at , g for hr at °c (hitachi cp wx ultracentrifuge). the resulting pellet was resuspended in μl of ten buffer and loaded onto a discontinuous sucrose gradient ( %, % and % sucrose (w/v) in ten buffer). the material was harvested from each sucrose interface and used for further analysis. osterhaus ad: a newly discovered human pneumovirus isolated from young children with respiratory tract disease epidemiology of human metapneumovirus the emergence of human metapneumovirus characterization of human metapneumovirus f protein-promoted membrane fusion: critical roles for proteolytic processing and low ph intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein respiratory syncytial virus and metapneumovirus. in fields virology analysis of the genomic sequence of a human metapneumovirus genetic diversity between human metapneumovirus subgroups antigenic and genetic variability of human metapneumoviruses metapneumoviruses in birds and humans human metapneumovirus detection in patients with severe acute respiratory syndrome analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time pcr detection of human metapneumovirus in respiratory secretions by reversetranscriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups immunofluorescence assay for detection of human metapneumovirusspecific antibodies by use of baculovirus-expressed fusion protein diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the north-east of england increased hydroxymethylglutaryl coenzyme a reductase activity during respiratory syncytial virus infection mediates actin dependent inter-cellular virus transmission the rsv f and g glycoproteins interact to form a complex on the surface of infected cells distribution of the attachment (g) glycoprotein and gm within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells pneumoviruses: the cell surface of lytically and persistently infected cells the human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro evidence for the interaction of the human metapneumovirus g and f proteins during virus-like particle formation respiratory syncytial virus assembly occurs in gm -rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin- role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus cooperativity of actin and microtubule elements during replication of respiratory syncytial virus ultrastructural analysis of the interaction between f-actin and respiratory syncytial virus during virus assembly protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein in virus particle assembly interactions between cellular actin and human respiratory syncytial virus (hrsv) functions of lipid rafts in biological membranes influenza viruses select ordered lipid domains during budding from the plasma membrane quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of f-actin and the raft-lipid microdomains in virus morphogenesis we acknowledge the national medical research council (singapore) for research support (nmrc/ / ). l.h. loo was a recipient of a nmrc-lee foundation scholarship. additional file : figure s . time course study of llc-mk cells infected with human metapneumovirus (hmpv). (a) llc-mk cells were infected with hmpv and at days ( dpi), days ( dpi) and days post-infection ( dpi) the cells were fixed and stained with anti-m. virus antigens were detected by immunofluorescence microscopy (anti-m). infected cells clusters are highlighted (white arrows) (x objective). (b) llc-mk cells were infected with hmpv and at dpi the cell monolayer was stained using anti-g and evans blue (eb) (to visualise the cell monolayer). hmpv filaments are indicated (white arrows).additional file : figure s . analysis of the surface topology of mock-infected and hmpv-infected llc-mk cells using scanning electron microscopy (sem). at days post-infection mock-infected and hmpv-infected llc-mk cells (grown on mm glass coverslips) were processed for sem as described previously [ , ] . briefly, the cells were incubated in the primary fixative ( %(v/v) glutaraldehyde in pbs, washed extensively in pbs and then incubated in the secondary fixative ( % osmium tetroxide). the cells were dehydrated using a - % (v/v) ethanol gradient and critical point-dried (polaron cpd) using ethanol. the cells were mounted on aluminium stubs and gold-coated. the processed cells were visualized with a quanta feg (fei) scanning electron microscope using appropriate machine settings (magnification x , ). the presence of microvilli (mv), virus filaments (vf) and spherical bodies (s) are highlighted.abbreviations hmpv: human metapneumovirus; rsv: respiratory syncytial virus; vlps: virus-like particles; ctx-b-af : cholera toxin b-subunit conjugated to af ; f protein: fusion protein; g protein: attachment protein; sem: scanning electron microscopy; feg: field emission gun; g-flag: g protein with a c-terminal flag tag; f-cmyc: f protein with a c-terminal cmyc tag. the authors declare that they have no competing interests. key: cord- - y d authors: li, zhiping; li, jinsong; zhang, yandong; li, lin; ma, limin; li, dan; gao, feng; xia, zhiping title: aerosolized avian influenza virus by laboratory manipulations date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: y d background: avian h n influenza viruses present a challenge in the laboratory environment, as they are difficult to collect from the air due to their small size and relatively low concentration. in an effort to generate effective methods of h n air removal and ensure the safety of laboratory personnel, this study was designed to investigate the characteristics of aerosolized h n produced by laboratory manipulations during research studies. results: normal laboratory procedures used to process the influenza virus were carried out independently and the amount of virus polluting the on-site atmosphere was measured. in particular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation, and experimental zoogenetic infection were performed. in addition, common accidents associated with each process were simulated, including breaking glass containers, syringe injection of influenza virus solution, and rupturing of centrifuge tubes. a micro-cluster sampling ambient air pollution collection device was used to collect air samples. the collected viruses were tested for activity by measuring their ability to induce hemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription pcr and ha test. the results showed that the air samples from the normal centrifugal group and the negative-control group were negative, while all other groups were positive for h n . conclusions: our findings suggest that there are numerous sources of aerosols in laboratory operations involving h n . thus, laboratory personnel should be aware of the exposure risk that accompanies routine procedures involved in h n processing and take proactive measures to prevent accidental infection and decrease the risk of virus aerosol leakage beyond the laboratory. the pandemic h n outbreak of and global threat of h n in recent years have been accompanied by a large amount of laboratory-based experimental research activity using purified viruses and infected tissues and animals. the majority of these studies have focused on either natural infections from the community or induced infections in the laboratory, with very few studies considering the infection risk or outcome of laboratory personnel handling the samples. the potential of an accidental laboratory-acquired infection is well-recognized among laboratory staff and researchers. in , meyer and eddie published the first report of laboratory infections due to the gram-negative bacteria brucella [ ] . in , sulkin and pike published a report that summarized laboratory-acquired infections due to viruses [ ] . since then, significant efforts have been made by the oversight committees of research institutes and governmental bodies to establish occupational and environmental safety guidelines to protect workers and the local community alike from laboratory-acquired infections; however, these infections have yet to be eradicated and many have been reported over the past eight decades [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the total number and relative frequency of bacterial laboratory-acquired infections has, in fact, declined dramatically over time [ ] [ ] [ ] . in contrast, the relative frequency of viral laboratory-acquired infections has increased by % [ ] . research into the underlying factors responsible for these infections have indicated that the main route of infection, for both bacteria and viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , is through mucous membranes that are contaminated by inhaling pathogens, not dissimilar from the natural route of infection [ ] . traditionally, the risk of laboratory infection has been minimized by simply practicing good laboratory practice (glp), which is otherwise necessary for reliability of the laboratory work itself. a detailed examination of the publically available information on all reported laboratory-acquired infections, however, indicated that~ % were not the result of overt "accidents"; it is, thus, likely that inhalation of aerosolized infectious particles that are liberated by normal laboratory techniques account for a large portion of laboratory-acquired infections. research into this theory has indicated that influenza virus transmission and infection can be achieved through aerosols [ ] . many of the routine procedures used to process influenza virus for laboratory research, such as centrifugation or mixing, have a high potential of producing aerosols [ ] , and the particle load of each has been estimated to be up to - μm. in addition, it is expected that larger particles will tend to fall out of the air and contaminate surfaces, by which individuals may be infected by contact or may transmit the particles to a secondary aerosol [ ] . fundamentally, aerosols are suspensions in the air of solid or liquid particles small enough that they will remain transmissible and airborne for a prolonged period of time [ ] . particles of μm or less increase the risk of establishing an infection upon airborne transmission, as they are remarkably capable of penetrating the physical cellular barrier of the respiratory tract and traveling all the way to the alveolar region. as with naturally-acquired infections, most individuals are not diagnosed before onset of symptoms, impeding the time to initiation of treatment [ ] . the a type influenza viruses are commonly spread by the airborne route in normal circumstances. accordingly, more research on the potential and character of aerosol spread of influenza virus has been carried out [ ] [ ] [ ] , and many studies have used experimental animal models of aerosol infection to mimic the natural process [ , , ] . however, less information is available on the features of laboratory-produced aerosolized influenza virus. the australian researcher, adrian gibbs, suggested that the a/h n flu virus currently circulating around the world was created in a laboratory. although the world health organization (who) eventually dismissed this theory, suspicion and panic were aroused in the general public about laboratory safety. there are many situations that may facilitate the spread of a pathogen from the laboratory, ranging from aerosols produced by routine procedures or misuse of laboratory equipment to uncontrollable natural disasters that impact the structural integrity of the laboratory, such as earthquake or fire. in order to regulate the potential of pathogen transmission from the laboratory, we must first gain a detailed understanding of the experimental operations that produce aerosols. to this end, this study was designed to monitor the presence of aerosolized h n virus produced by normal procedures used to process the virus for experimental research and by the most frequently associated "accidents" for each, such as container breakage and accidental subcutaneous injection. this information will help to guide future experimental practice standards to ensure the safety of laboratories and laboratory personnel, thereby increasing community confidence in laboratory bio-safety. the prototype sampling device, shown in figure , consisted of a controller and six pumps with agi- or andersen impingers of a wireless networking technology. this instrument can collect several samples of aerosols simultaneously, or can be set to have one of the six pumps operate independently. we used liquid impingers, which rely on inertial collection mechanisms to collect aerosolized particles [ ] and were situated as described in the materials and methods. the sampler outlet was figure micro-cluster sampling ambient air pollution collection device. the device consists of a controller and six pumps with agi- or andersen (a). the device relies on the liquid impactor approach to capture airborne viruses (b). red, thin lines and arrows represent the airflow into the sampler. blue, thick arrows represent airflow out of the sampler (c). the cartoon is a simplified representation. situated above the nozzle outlet; thus, there was a sharp turn in flow streamlines at the nozzle outlet, just above the liquid surface. particles with high inertia cannot follow sharp turns in streamlines and will impact and penetrate the liquid surface after exiting the nozzle. at the start of each group experiment, the sampler was operated for minutes to obtain baseline readings of the laboratory atmosphere. collection of aerosols from the seven groups of experimental procedures and controls followed the steps detailed in the materials and methods section. the processing time was min for each group, except for group i (time was h), and the air sampling distance was cm from the working materials. statistics of the group temperature and relative humidity for each of the data collection processes, including operations and control of the collection, were measured automatically by the aerosol collector. none of the aerosol samples collected from any group at the time directly prior to processing of the experiment had detectable levels of h n , as evidenced by negative reverse transcription (rt)-pcr and ha text results. all liquid aerosols collected from the experimental procedure of group iii were also negative for h n (table , figure ). the aerosols collected during the experimental procedures of all other six groups were positive for h n . the aerosols from each group taken after disinfection were negative ( table ) . our experiment was carried out in a negative pressure isolation unit with mechanical arm ( figure ). the three groups of procedures with simulated accidents (broken glass containers with influenza virus suspension, syringe-ejected influenza virus suspension, and centrifuge tube rupture) all produced aerosols. the controls for each were all negative. all of the aerosols that were produced contained h n that was detectable by rt-pcr and ha text (table , figure ). the field of laboratory-acquired viral infections has progressed since sulkin and pike's seminal report in [ ] , but the problem has yet to be completely eliminated. the paucity of experimental data is an important reason for this. our study, presented herein, provides the first evidence of laboratory procedures that generate viral contamination that can potentially infect laboratory personnel. in fact, all of the studies performed to date by others to determine whether laboratory studies of pathogens, including viruses, bacteria and fungi, could generate infective aerosols have been based on statistical analysis of actual infections. the influenza viruses present a particular challenge to monitoring in the lab by traditional methods, since their small size and low concentration make them difficult to collect and detect from the air. most of the bioaerosol samplers currently on the market are not suitable for collection of viruses [ ] . furthermore, effective detectors need to be able to conveniently collect air samples at various time points over the course of an entire experiment since virus-containing aerosols may be generated by any number of steps in the process and then fall from the air prior to an end-of-procedure sampling time point. the currently available samplers typically run for short periods of time (minutes), making it difficult to capture large volumes or integrate sample collection over time. we designed a collector terminal with six air pollution sampling probes that are able to continuously sample the air for ≥ h; in addition, this device is portable and can be easily moved around the laboratory to locales where different steps of the experiment are performed. the feature of remote control allows for simultaneous or sequential collection in different locales from two or more of the probes. in the study described herein, to test the capability of such a detector, the aerosols collected during the experiment reflected the virus produced in most operations; this study was not designed to clearly delineate a complete picture of all potential applications of this device. measuring infectious virus from air samples is logistically difficult. few researchers have reported airborne influenza virus from the laboratory, and even fewer have detected the infectious capacity of influenza viruses sampled from air. traditional methods of determining the presence of virus in an aerosol include directly applying the concentrated aerosol to infect a host cell, or as template in rt-pcr to detect virus-specific genes. one of the most commonly used methods is rt-pcr because of its remarkable sensitivity and specificity; however, rt-pcr is unable to determine whether the detected virus is infectious. in our study, we combined experimental approaches that would determine both the concentration of virus in aerosols and the activity of those viruses. by exploiting the functional character of the hemagglutinin of influenza virus, we were able to estimate the concentration of influenza virus in aerosols according to the amount of adsorption that occurred with red blood cells (rbcs). by also using the aerosols to directly inoculate chick embryos, we were able to determine the ability of the aerosolized virus to proliferate in vivo, indicating the infectivity of the contaminating pathogens. future studies will aim to determine the feasibility of this approach for quantifying virus in the aerosol. this study, to our knowledge, represents the first successful attempt to directly detect influenza viruscontaining aerosols generated by routine laboratory procedures. our results provide evidence that many of the laboratory techniques used to process influenza virus for experimental analysis produce aerosols and, thereby, represent significant risks of infection to laboratory personnel and potential spread beyond the laboratory. working in the laboratory is inevitably dangerous, but nearly all risks can be sufficiently minimized by glp and careful monitoring of risk factors, such as presence of pathogen-containing aerosols. according to the results of this study, we plan to extend our investigations to emergency operating procedures that follow laboratory accidents with virus samples and to generate more effective strategies to prevent laboratory-generated aerosols and laboratory-acquired infections or spread. in summary, our findings demonstrated that many of the laboratory techniques used to process influenza virus produce aerosols, either through normal operations or common accidents associated with each process. our results have great value and implications towards biosafety and future strategies to evaluate risks of experimental virology to laboratory workers and the general public. purified avian influenza virus (a/chicken/jilin/ / (h n ), genbank: ay ~ay ) was obtained from our laboratory stock, and diluted - in phosphate buffered saline (pbs, . mol/l) supplemented with free calcium and magnesium. aliquots of this stock solution were stored at − °c until use. prior to experimentation, virus was propagated by infection in chicken eggs, and yield was determined to be . log % egg infection doses per milliliter (eid /ml). all the experiments on animals were approved by the ethics committee aerosol collection was carried out with the micro-cluster sampling device that was designed by the academy of military medical sciences (beijing, china). airborne biological particles were gathered using liquid impingers (all glass impinger , agi ; ace glass inc., vineland, nj, usa) that rely on inertial collection mechanisms. the flow rate was . l min - , when the pressure drop across the orifice was cm hg. the tip of the capillary stem was situated mm from the flask bottom; therefore, when filled with ml of liquid, the nozzle outlet was mm above the resting liquid surface. airborne biological particles drawn into the mm diameter nozzle and down the capillary stem, then impacted and penetrated the liquid surface [ ] . the adhesion properties involving liquid and airborne particles were exploited by this technology to capture the microorganisms [ ] . all assays were conducted in a biosafety level setting. the containment laboratory -biosafety level was designed for work with risk group microorganisms in large volumes and risk group microorganisms at high concentrations. in addition, the biosafety level containment laboratory was equipped with a negative pressure isolation unit, which was used in our study for simulating accidents during laboratory procedures. normal laboratory procedures were carried out under controlled conditions for the purpose of monitoring the amount and character of aerosol produced. in group i (zootomy), chickens diagnosed with aiv upon autopsy were sectioned and the excised tissues frozen at − °c. the total collection time was h. disinfection of the anatomical units after collection and air sampling took an additional h. control air samples were taken in the same units one day later. this process was repeated a total of six times, and six chickens were necropsied. in group ii (grinding), the frozen lung tissues were thawed, ground in a mortar, and transferred to a centrifuge tube for storage at °c. aerosol collections were carried out during grinding (at min into the procedure) and min after the homogenate was collected, for a total of min. after disinfection of the area, air samples were immediately collected. this entire process was repeated six times. in group iii (centrifugation) the tissue homogenates were centrifuged at rpm for min at °c. air samples were collected from the point of initial handling of the centrifuge tube to the centrifuge to the end of the spin when the centrifuge instrument was opened and the tube removed. to balance the centrifuge for each spin, the six homogenates were processed in groups of two, so that the experiment was repeated three times. air sampling was performed after disinfection for use as the control. collection times were min. in group iv (pipetting), the supernatant resulting from the centrifugation step was transferred by a pipettor to a new tube, using blow-out and pull-in processes a total of times to mix each sample. to ensure continuity of the experimental integrity, the pipetting experiment was carried out in another similar laboratory room that did not communicate with the centrifuge room. the same air sampling procedure was carried out for min during the experiment and after disinfection. similar to group iii, the procedures of group iv were repeated three times. in group v (magnetic stirring), the viruscontaining supernatant samples were diluted with pbs (ph . ) in a -fold series. this occurred by taking ml of supernatant and adding to pbs in a ml beaker with a stir bar that was placed on an active magnetic stirrer. air sampling occurred throughout the entire process and after disinfection of the space, three times at min each. in group vi (egg inoculation), chick embryos (about -days-old) were inoculated with . ml diluted virus sample ( -fold diluted virus stock solution) per embryo. air samples were collected throughout the process, including the time when the virus was drawn into the syringe, air bubbles were removed from the syringe, chick embryos were injected, and sealing wax was applied. a total of chicken embryos were inoculated in each air monitoring experiment. air samples were collected throughout and after disinfection, taking min. the procedure was repeated three times, requiring a total of chick embryos to be inoculated. in group vii (zoogenetic infection), five chickens ( -days-old) was intranasally administered virus at a dose of . ml per chicken. the zoogenetic infection experiments were repeated three times. air sampling was carried out at intervals of min each, and after disinfection. laboratory simulation of accidents that most frequently occur during the routine processing of influenza viruses were carried out inside a negative pressure isolation unit equipped with a simple robotic arm and operating gloves. in accident group i (broken glass containers), glass containers holding influenza virus suspensions were dropped in a free-fall and the broken pieces collected as in a routine clean-up procedure. in accident group ii (syringe-ejected influenza virus suspension), ml of the virus dilution ( -fold diluted virus stock solution) was drawn into a disposable syringe, and the plunger was depressed to spray out a small amount of the virus suspension into the air. in accident group iii (centrifuge tube rupture), empty microcentrifuge tubes were centrifuged at inappropriate speeds to produce cracked tubes. then, ml of virus solution was added to the tube and the tube was closed and manually squeezed until the tube ruptured and the liquid splashed out, at which point air samples were collected. all of the processes for the three groups were repeated five times. for each, air samples were collected before the procedure for use as the control. the collection time was min. after virus propagation in chicken eggs, the allantoic fluid was collected and processed according to standard procedures for detections of aiv by rt-pcr and ha text, both detections were repeated twice. the sensitivity of the rt-pcr procedure to detect aiv was evaluated by using a -fold diluted virus series; aiv was detectable by this method up to [ ] dilution of the virus solution ( figure ). the processing of samples collected by the air monitor was based on well-known hemagglutination properties of h n [ ] . briefly, the virus in each sample would bind to rbcs, causing an agglutination effect that was proportional to the virus concentration. in addition, the samples were used to inoculate -day-old embrocated chicken eggs. eggs were incubated for h at °c and then chilled overnight at °c. the allantoic fluid from each egg was collected separately and detected by standard rt-pcr. for long-term storage, chicken rbcs (crbcs) were treated with formaldehyde. treatment of erythrocytes. chicken blood was divided into several tubes and centrifuged at rpm for min; the resultant supernatant was carefully removed by pipetting and mixed with sterile pbs (ph . ). three pbs washing steps by centrifugation were carried out. finally, the erythrocyte sediment was diluted with pbs at a : (vol/vol) ratio and . % glutaraldehyde was added at a : (vol/vol) ratio and the solution was oscillated at room temperature for min. the treated erythrocytes were washed four times with pbs by centrifuging ( rpm for min each), and finally resuspended in pbs at % (treated erythrocytes/ volume of pbs) and stored at °c until use. treatment of sample. samples collected from experimental groups and their respective controls were incubated on ice to chill. then, treated erythrocytes were added at a : . (vol/vol) ratio. adsorption was allowed to proceed for h at °c, during which time the sample was intermittently mixed by inverting the tubes several times. after the incubation, samples were centrifuged at rpm for min at °c, and the supernatant was discarded. the sediment was resuspended with ml pbs on ice and recentrifuged at rpm for min at °c, after which the supernatant was discarded. the sediment was then resuspended in a mixture of . ml amb-antibacterial media (supplemented with penicillin and streptomycin, u/ml each) and . ml pbs. the mixture was incubated for h at °c with intermittent mixing by inverting the tubes several times. the entire volume ( . ml) of each sample was then used to inoculate a -day-old embryonated chicken egg. the inoculated eggs were incubated for h at °c and then chilled overnight at °c. the allantoic fluid from each egg was collected, except in the cases where the embryo had died within h after inoculation. a standard rt-pcr test was performed to confirm the presence of the virus in allantoic fluid. a total of . ml of the allantoic fluid was used for rna extraction by the qiagen rneasy mini kit (cat no. ) and then applied to a reaction mix using reagents from the one-step rt-pcr amplification mini kit (qiagen cat no. ), as follows: initial heating step, °c for min; reverse transcription, °c for min; initial pcr activation step, °c for min; cycles of amplification (denaturation, °c for min; annealing, °c for min; extension, °c for min); final extension, °c. genespecific primers for the np gene of h n were: np-forward, ′-gcattgtctccgaagaaataag- ′ and np-reverse, ′-cagatact gggchataagrac- ′. the expected length of the amplicon was bp. the allantoic fluid (positive by rt-pcr) was detectable by hemagglutination test that performed to confirm the presence of the virus. hemagglutination test was performed in -wells microtiter plates with % crbcs. laboratory infections due to brucella viral infections contracted in the laboratory survey of laboratory-acquired infections aerosol hazards from some clinical laboratory apparatus risk of human immunodeficiency virus infection among laboratory workers figure rt-pcr analyses of virus adsorb-proliferation sensitivity. lanes: negative; dna markers - rt-pcr results of -fold diluted series of virus solution rift valley fever: a report of cases of laboratory infection western equine encephalomyelitis in a laboratory worker louping ill in man laboratory safety in research with infectious aerosols laboratory-associated infections and biosafety overview of the epidemiology of laboratoryacquired infections viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects laboratory-associated infections. summary and analysis of cases pathology of experimental inhalation anthrax in the rhesus monkey the pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation studies on respiratory infection. iii. experiments with brucella suis. hyg camb molecular pathology in the lungs of severe acute respiratory syndrome patients. immunopath and infect dis an experimental model to evaluate the role of transport vehicles as a source of transmission of porcine reproductive and respiratory syndrome virus to susceptible pigs quantification of airborne influenza and avian influenza virus in a wet poultry market using a filter/real-time qpcr method inefficient transmission of h n influenza viruses in a ferret contact model epidemiology and control of virus infections in the laboratory aerosol technology aerosol transmission of influenza a virus: a review of new studies pathogenesis by aerosol infectious diseases: biological weapons defense. infectious diseases and counterterrorism review of aerosol transmission of influenza a virus transmission of influenza a in human beings experimental transmission of influenza virus infection in mice. iv. relationship of transmissibility of different strains of virus and recovery of airborne virus in the environment of infector mice influenza a virus transmission: contributing factors and clinical implications transmission of influenza virus via aerosols and fomites in the guinea pig model collection efficiencies for aerosol samplers for viruscontaining aerosols airborne viruses airborne influenza virus detection with four aerosol samplers using molecular and infectivity assays: considerations for a new infectious virus aerosol sampler bioaerosol sampling for the detection of aerosolized influenza virus the agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus aerosolized avian influenza virus by laboratory manipulations the authors would like to thank the scientific editing department for editorial assistance with this manuscript. this work was supported by a grant from the national science and technology major project (no. zx - ). finally, we are grateful to pro. li from the academy of military medical sciences for experimental guidance. the opinions expressed in this paper are solely those of the authors and do not necessarily reflect the views of the funding agency. we thank medjaden bioscience limited for assisting in the preparation of this manuscript. rt-pcr: reverse-transcript polymerase chain reaction; ha: hemagglutination; glp: good laboratory practice; who: world health organization; agi : all glass impinger ; crbc: chicken red blood cell. the authors declare that they have no competing interests. key: cord- -qjyooq authors: king, chwan-chuen; chao, day-yu; chien, li-jung; chang, gwong-jen j; lin, ting-hsiang; wu, yin-chang; huang, jyh-hsiung title: comparative analysis of full genomic sequences among different genotypes of dengue virus type date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qjyooq background: although the previous study demonstrated the envelope protein of dengue viruses is under purifying selection pressure, little is known about the genetic differences of full-length viral genomes of denv- . in our study, complete genomic sequencing of denv- strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the denv genome other than within the e gene were also analyzed. results: using maximum likelihood and bayesian approaches, our phylogenetic analysis revealed that the taiwan's indigenous denv- isolated from and dengue/dhf epidemics and one sporadic case were of the three different genotypes – i, ii, and iii, each associated with denv- circulating in indonesia, thailand and sri lanka, respectively. sequence diversity and selection pressure of different genomic regions among denv- different genotypes was further examined to understand the global denv- evolution. the highest nucleotide sequence diversity among the fully sequenced denv- strains was found in the nonstructural protein a (mean ± sd: . ± . ) and envelope protein gene regions (mean ± sd: . ± . ). further analysis found that positive selection pressure of denv- may occur in the non-structural protein gene region and the positive selection site was detected at position of the ns gene. conclusion: our study confirmed that the envelope protein is under purifying selection pressure although it presented higher sequence diversity. the detection of positive selection pressure in the non-structural protein along genotype ii indicated that denv- originated from southeast asia needs to monitor the emergence of denv strains with epidemic potential for better epidemic prevention and vaccine development. dengue fever (df) and its more severe forms, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss), have emerged as major public health problems in tropical and subtropical areas [ , ] . infection with dengue viruses (denv), which are maintained in a human-mosquito transmission cycle involving primarily aedes aegypti and aedes albopictus, can result in various clinical manifestations ranging from asymptomatic to df, dhf, dss and death [ ] . the occurrences of dengue epidemics in the past years have been characterized by the rising incidence rates of infection and continuous expansion in geographic distribution of dhf epidemics [ ] . importantly, the epidemics of dhf have become progressively larger in the last years in many dengue endemic countries [ ] . the increasingly widespread distribution and the rising incidence of df and dhf are related to increased distribution of a. aegypti, global urbanization and rapid and frequent international travel. epidemiological analysis reveals that some denv strains are associated with mild epidemics with low occurrences of dhf cases and inefficient virus transmission, whereas others are more likely to cause severe epidemics with high incidence of dhf/dss and rapid virus transmission [ , ] . the large dhf epidemics in indonesia in the s and sri lanka after provided evidence supporting this phenomenon [ , ] . dengue virus serotype (denv- ) re-appeared in latin americain after its absence for seventeen years. the virus was detected initially in panama and soon dispersed throughout central and south america during the following years [ , ] . this introduction coincided with an increased number of dhf cases in this region. although the genotype originating in southeast asia has been postulated as the major cause of the increased virulence, the molecular marker associated with a difference in virulence among genotypes at the fullgenomic level is still largely unknown. dengue is caused by four antigenically related but genetically distinct viruses (denv- , - , - and - ) belonging to the genus flavivirus, family flaviviridae [ ] . denv is a single stranded, positive-sense rna virus, approximately , nucleotides in length. the genome contains a single open reading frame (orf) that encodes a polyprotein, which is co-and post-translationally processed to produce three structural proteins, including capsid (c), pre-membrane (prm) and envelope (e), and seven nonstructural (ns) proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ , ] . a considerable number of studies have revealed that each serotype of denv is composed of phylogenetically distinct clusters that have been classified into "genotypes" or "subtypes," and each genotype is also composed of phylogenetically distinct "groups" or "clades." a previous study has classified denv- strains into four genotypes based on limited numbers of nucleic acid sequences from the prm and e protein genes [ ] ; denv- strains have also been re-classified into five genotypes [ ] . growing evidence suggests the existence of denv strains with different epidemic potentials. this evidence is supported by the following observations: ( ) the differences in fitness among various genotypes of denv- reflect their different replication capabilities in human monocytes and dendritic cells [ ] ; ( ) around , clade replacement among denv- genotype ii containing isolates from thailand was associated with changing serotype prevalence and incidence of dhf epidemics [ ] ; and ( ) sudden changes in the genotype of denv at a single locality have been observed that appeared to originate from the genetic bottleneck of a large viral population [ , ] . this sudden genotype replacement has been associated with more severe dhf epidemics in indonesia and sri lanka [ , ] . however, most of these studies involved the e gene alone. this raises an important question: is the introduction of different denv genotypes in disparate geographical locations a result of sequence differences outside of the e gene altering their epidemic potential, or it is simply a stochastic event in viral evolution? dengue epidemics in taiwan are usually initiated by imported index cases (king et al., ) . the re-emergence of dengue outbreaks in taiwan started when denv- was re-introduced into the off-islet of hsiao-liu-chiu in . in - , another large-scale denv- outbreak occurred in kaohsiung and pingtung in southern taiwan [ ] . although denv- was detected sporadically from imported index cases, no denv- -related epidemic occurred until dhf cases were confirmed in kaohsiung in and dhf cases in tainan in [ ] . taiwan neighbors many southeast asian countries and more than , travelers visit these adjacent countries annually. the surveillance system implemented by the center for disease control in taiwan (taiwan-cdc) routinely detects many imported dengue cases each year. thus, taiwan is an ideal place to study the evolution and dispersion of denv that may have different epidemic potential, particularly in the and dhf epidemics in taiwan that coincided with the dhf epidemics in southeast asian countries [ ] . complete genomic sequencing of denv- strains collected from different geographical locations and isolation years offers the opportunity to understand the genetic stasis and possible selection pressure sites in the denv genome other than within the e gene. the blood samples of suspected dengue patients, obtained from the sentinel hospitals/clinics located in tainan, kaohsiung and pingtung in southern taiwan, were sent to the infectious disease epidemiology laboratory at national taiwan university (ntu) and taiwan-cdc for laboratory confirmation. the study protocol was approved by the college of public health research human subject ethics review committee at ntu. a suspected and confirmed dengue case was defined as previously described and confirmed by both laboratories [ , ] . imported and indigenous dengue cases were defined based on the patients' travel history to dengueendemic or -epidemic countries within - days before the onset of the disease. due to few denv- epidemics and limited denv- isolates identified before in taiwan, we focused our study on comparing the sequences of different denv- isolates in and considering various epidemiological characteristics, including temporal, geographical and host factors. six denv- isolates were selected for full-length sequencing: ( ) an isolate from the imported denv- infected case in ; ( ) an isolate from the indigenous df and dhf cases during the epidemic in tainan, taiwan; ( ) the isolate from a geographical location in tainan other than the epidemic area; ( ) an isolate from the same geographical location as the tainan's epidemic but in ; and ( ) an isolate from indoor mosquitoes during the dengue/dhf epidemic in tainan. the epidemiological characteristics of these six denv- isolates are summarized in table , and their genebank accession numbers are dq -dq . in addition to the - denv- strains, four local isolates obtained from taiwan during previous years, kindly provided by taiwan-cdc, were also used for comparison, including four strains isolated from indigenous df patients during the - epidemic in kaohsiung [ twkh (accession no.: dq ), twkh (accession no.: dq ), twkh (accession no.: dq ), tw (accession no.: dq )]. isolate tw with low passage history (two passages in c / cells) was subjected to full-length genomic sequencing together with the above six isolates from - , constituting seven full-length denv- sequences from taiwan. the remaining three denv- isolates were sequenced only from the ' ncr to the cooh-terminus of the e gene region for phylogenetic analysis. acute-phase serum or plasma samples collected from the dengue patients within seven days after the onset of fever were used for both virus isolation and molecular diagnosis [ , ] . molecular diagnosis by reverse transcriptase polymerase chain reaction (rt-pcr) amplification and subsequent nucleic acid sequencing was performed as previously described, and a complete list of the pcr and sequencing primers utilized is available upon request [ ] . the rna genomic ' and ' terminal nucleotide sequences were not confirmed independently and were assumed to be of the same length and sequence as the prototype strain h in this study. a total of complete genomic sequences of denv- strains and one denv- strain a (genbank accession number ab ) were aligned using the multiple sequences alignment clustalx [ ] . these sequences were further combined with all available sequences of the complete e gene or the complete prm and partial e genes (to nucleotide position of the e gene) of denv- deposited in the genbank database at the national center for biotechnology information (ncbi). therefore, the complete e gene ( nt) dataset consisting of a total of sequences and the prm and partial e gene ( nt) dataset of a total of sequences were used for phylogenetic analysis. a complete list of the sequences along with associated epidemiological information is available upon request. the percentage of sequence similarities and differences were calculated using bioedit v . program [ ] . pairwise comparisons of both nucleotide and amino acid sequences of denv- isolates were performed using the program mega v . (molecular evolutionary genetics analysis, pennsylvania state university, pa) to determine the mean and range of the proportional difference (p-distance) [ ] . the model of nucleotide substitution that best described denv- sequence evolution was identified using the program modeltest . [ ] . the resulting most complex gtr+i+Γ substitution model (general time reversible model, gtr, a proportion of sites modeled as invariant, i, variation in rates among sites modeled using the gamma distribution, Ã) was selected to be the best fit to the data using the hierarchical likelihood ratio tests (hlrts) and akaine information criterion (aic). the estimated parameter values from this model were as follows: relative substitution rates among nucleotides were a ↔ c = . , a ↔ g = . , a ↔ t = . , c ↔ g = . , c ↔ t = . , g ↔ t = . ; proportion of invariable sites (i) was . ; gamma distribution of among-site rate variation (Ã) was . ; and estimated base composition of a = . , c = . , g = . , and t = . . a maximum likelihood (ml) tree using these parameter settings was estimated using the dnaml in phylip v . package [ ] . bootstrap analysis with , re-samplings was used to determine confidence values for groupings within the phylogenetic tree. in addition, a posterior probability distribution tree, generated by implementing the recently developed bayesian hierarchical phylogenetic model utilizing a metropolis-coupled monte carlo markov chains (mc) algorithm in the mrbayes program (version . , [ ] ) was compared with the evolutionary tree of denv- generated by the ml method. indeed, the bayesian approaches for constructing phylogenetics have several advantages. first, the primary analysis often provides faster estimates of the tree and measurements than the estimates obtained using ml bootstrapping techniques. secondly, bayesian model selection offers advantages over likelihood methods in that the competing evolutionary hypotheses need not to be nested, and it does not rely on standard likelihood assumptions. in other words, the starting trees in bayesian method are randomly chosen, and multiple runs of the same dataset are generally made with different starting trees to check convergence of the process. the programs' default settings for prior probability were used in our analysis. bayesian markov chain monte carlo (bmcmc) processes, considering the heterogeneity in the evolutionary process and thus incorporating a discrete gamma distribution of four classes of substitution rates across mutation sites, were run for , generations. output trees were sampled every generations but the first , trees were discarded before the process reached the convergence state. the resulting trees were rooted using a denv- strain a isolate as described. to analyze the selection pressure in denv- , the codeml program from the paml package was employed by implementing a maximum-likelihood method. this method presents major advantages over simpler pairwise comparisons in considering the transition/transversion rate bias, non-uniform codon usage, and phylogenetic relationships among the sequences [ ] . positive selection at a small number of codons can be detected by comparing various models of codon evolution which differ in how the rates of synonymous (ds) and nonsynonymous (dn) substitutions (denoted as ω) are treated among codons or within lineages using likelihood ratio tests. to analyze selection pressures at individual codons, we compared the m and m model. in the m model, categories were assigned and estimated from the data, which specified only neutral evolution; however, the m model allowed positive selection by add-ing an th codon category at which dn/ds can exceed . . to examine selection pressures along the lineages, the free ratio model, which allows certain lineages to have ω ratios different from the background, was implemented in the m model. additionally, parameters involving the incorporation of classes of codons where ω > were used by comparing the value of the likelihood from m , in which the specified neutral evolution of ω is constrained to be equal to or less than at all codons among all lineages. the comparison was again assessed using the likelihood ratio test. if positive selection was found, the bayesian method was applied to identify the specific codon that may have been subjected to positive selection pressure. we have determined the complete nucleotide sequences ( , nucleotides in length with an orf of , amino acids) of the seven different denv- strains from taiwan ( table ). the percentages of nucleotide and amino acid identities of the entire orf among these strains, compared with the prototype denv- strain h isolated in the philippines in , are shown in table . the indigenous denv- isolates from the epidemic area in tainan city ( tw and tw ) and from the sporadic case in pingtung ( tw ) displayed the highest similarity, with . % sequence identity in both nucleotide and amino acid sequences. the imported tw strain showed slightly lower nucleotide and amino acid sequence identity ( %) relative to these indigenous taiwanese denv- isolates. the denv- isolates of taiwan from years other than , including the kaoshiung tw and the tainan tw strains, showed higher sequence diversity compared with the denv- taiwan isolates ( % nucleotide and amino acid sequence identity), which suggested that they might have originated from different countries. further phylogenetic analysis revealed that these viruses belong to different genotypes (genotype i and iii; see the section ''phylogenetic analysis of denv- '' for details). compared to the prototype strain h , several unique amino acid substitutions that serve as unique signature sites for each genotype were found within the full genomic sequences of the selected denv- isolates from taiwan or other countries and are listed by the order of the gene in table . among those, several substitutions changed the polarity, charges, or hydrophobicity of these amino acids, which were present only in genotype iii of denv- , including the change from threonine (t) to alanine (a) at position of the c region, leucine (l) to histidine (h) at position of the prm region, l to t at position of the e region, isoleucine (i) to t at position of the ns region, and lysine (k) to t and aspartic acid (d) to asparagine (n) at positions and of the ns region. similar signature sites experiencing amino acid property alterations in genotype ii included a change from t to a at position of the prm region, l to serine (s) at position of the ns region, and a to t at position of the ns a region. thus, our data suggested that different genotypes of denv- experience different amino acid changes at both structural and non-structural genes, and the sites of these substitutions could serve as signature sites for genotype identification. the phylogenetic trees of denv- were constructed from the two different nucleic acid dataset alignments: ( ) partial sequences of the prm and e gene region (prm/e) from isolates obtained from taiwan and sequences available from genbank; ( ) complete e gene sequences including isolates from both taiwan and genbank. the trees derived from the maximum likelihood method the upper-right matrix corresponds to nucleotide sequences and the lower-left matrix to the amino acid sequences. geno v v i i i i ii ii ii ii ii iii iii iii t e v i i i i v i i i i l f f f f ns b v i i i i l and the bayesian method based on both datasets were very similar to each other. thus, only the posterior probability tree derived from the bayesian method based on the complete e gene sequences is shown (fig ) . the denv- strains isolated in taiwan with the lack of full-length sequences of viruses belonging to old american genotype iv, only four denv- genotypes, including representatives of genotype i ( tw ), genotype ii ( tw ), genotype iii ( tw ) and genotype v (h ) were compared. the sequence divergences in nucleotide and amino acid were calculated as the p-distance by adjusting the lengths of different genes [ , ] . the highest nucleotide diversity was found in the ns a gene (mean ± sd: . ± . ), followed by the e gene (mean ± sd: . ± . ). similar results were observed for amino acid diversity, which was also the highest in the capsid gene (mean ± sd: . ± . ), followed by the ns a gene (mean ± sd: . ± . ) ( table ) . to determine whether higher sequence diversity in certain genes could be the result of natural selection pressures, we implemented the m and m selection models to determine whether positive selection pressure among all codons from the full-length denv- sequences could be detected by using the codeml program from paml [ ] . the results suggested that both structural and non-structural genes of denv- were under neutral selection. although the e gene showed positive selection (ω = . ) with statistical significance (p = . ) when using the larger dataset with sequences, no specific site with positive selection could be detected. to further examine the selection pressure along the lineage, genotype i, ii, iii and v, based on the phylogenetic tree of the full-length sequences (fig ) , were examined separately using the m model. the results are summarized in table . although there were positive selection pressures detected in the c and ns b genes of genotype i, and in the e, ns and ns genes of genotype ii, only the ns gene of genotype ii showed statistically significant positive selection pressure. furthermore, positive selection was detected at position of the ns gene (substitution of s for l). changes occurring in the ' ncr and ' ncr were examined among the denv- viruses isolated in taiwan and other countries. in the ' ncr, positions , , and had nucleotide changes that were distinguishable for the specific genotype. among them, a g to a change at position was frequently seen in genotype i, a c to t change at position and a g to a change at position were observed only in genotype ii, and an a to g change at position was present in genotype iii. interestingly, the bayesian hierarchical consensus tree showing the phylogenetic relationships between denv- genotypes is based on the complete e gene sequences ( bp) from the denv- strains sampled globally figure the there was consistently an additional -nucleotide sequence, agtgaaaaaga, inserted in the ' ncr close to the end of the open-reading frame (orf) of the denv- strains isolated in recent years, compared to the prototype strain h . in the ' ncr, nucleotide changes at position , , and (nucleotide numbering beginning at '-terminus of ' ncr after the stop codon) were observed from the strains circulating recently, which differed from the strain h . however, none of these changes had any effect on the predicted secondary structure of the ' ncr rna (data not shown). the putative genome cyclization sequence ucaauaug, located between nucleotides and of the c gene, was conserved in all denv- viruses. viral sequence comparisons among isolates from dengue epidemics of different disease severities may provide valuable information regarding the molecular basis of the epidemic potential of the virus. denv- re-appeared in in taiwan and caused the df/dhf epidemic in tainan city after its first introduction in [ ] . this stimulates a great interest in understanding the molecular relationship of denv- isolates in taiwan during interepidemic periods and in comparing them with the strains circulating globally to understand evolutionary trends and geographical expansions. here, we confirmed that the dengue epidemics in taiwan were strongly associated with the globally circulating denv- due to constant introduction of viruses from southeast asia by taiwanese travelers. our data demonstrates the sequence diversity among the full-genomic sequences of denv- and the positive selection pressures exerted in different lineages (i.e. genotypes) at sites in denv- non-structural genes. since most taiwan dengue epidemics were initiated by the introduction of virus from imported cases [ ] , phylogenetic analysis provides essential information to understand the history and origin of all taiwan denv- isolates originating in other countries (fig. ) . the high nucleotide sequence identity (> . %) among the strains isolated in indicates that they were from a single origin and further spread to different townships, such as pingtung (id# tw ). the only imported denv- isolated from a traveler who had recently visited indonesia was more closely associated with the genotype ii isolates from myanmar and older isolates from thailand. this virus differed from the virus isolated during the tainan outbreak, which might suggest that multiple genotypes of denv- circulated in indonesia. this observation is consistent with a previous study indicating that at least two subtypes of denv- were present in indonesia [ ] . the phylogenetic analysis also suggested that a single isolate (id# tw ) from the same location as the epidemic was grouped together with the genotype iii sri lanka isolates. additional denv- isolates from the first denv- -caused dhf outbreak in taiwan ( ) ( ) were grouped into genotype i. all these results implicated that repeated introductions of different genotypes of denv- into taiwan since were important causes of dengue epidemics, and that denv- was not endemic in taiwan. this situation may be similar in the subtropical region of china. our country initiated airport fever screening during the severe acute respiratory syndrome (sars) outbreak in - , and it successfully identified confirmed, imported dengue cases [ ] . airport fever screening can thus quickly identify imported dengue cases, and may prevent a significant number of dengue outbreaks that would have been initiated by imported index cases. however, its cost-effectiveness in preventing any dengue epidemics in taiwan will need to be evaluated in the future. with different denv- genotypes imported into taiwan from southeast asia and other parts of the world, this virus collection provides an excellent opportunity to examine the sequence diversity of different genes of the full-length denv- viral rna genome for genotypes other than genotype iv. the highest p-distance of nucleotide diversity of the full-length genomes occurred for the ns a gene ( . % ± . %), followed by the e gene ( . % ± . %). in contrast, the highest p-distance of amino acid diversity of the full-length genomes occurred for the cap- the maximum likelihood phylogenetic tree shown here is based on the complete genomic sequences of denv- strains available from genbank figure the maximum likelihood phylogenetic tree shown here is based on the complete genomic sequences of denv- strains available from genbank. the tree was rooted using denv- strain a (genbank accession number: ab ) as the outgroup. the major amino acid changes along lineages within genotype i and ii are also labeled. taiwan denv- isolates are marked with a star. sid gene ( . % ± . %), followed by the ns a gene ( . % ± . %). this observation is consistent with the previous analysis in denv- , denv- and denv- [ , ] , although the precise cause of the increased rate of amino acid change in the ns a gene is unknown. a similar observation could also be made while analyzing the full genomic sequences of west nile virus (wnv) isolated from different animal species [ ] . the flavivirus ns a, a protein important for viral replication and particle formation [ ] , is cleaved by viral serine protease. a mutation at the basic p cleavage site residue in ns a blocks this processing event and is lethal for virus production while still allowing rna replication [ , ] . furthermore, this basic residue in ns a and an acidic residue in ns are important determinants for virus assembly and/or release [ ] . although the relative high sequence diversity of the ns a gene of denv- may be due to the lesser structural constraint required for ns a, it is possible that positive selection pressures may be exerted on this gene. especially in light of recent studies, ns a together with ns b and ns a were identified as dengue virus-encoded proteins that could antagonize the interferon (ifn) response during viral infection [ , ] . our analysis didn't detect any selection pressure exerted on the ns a gene probably due to the small sample size; future studies will be needed to focus the selection pressure analysis on non-structural proteins and denv evolution. several evolutionally conserved amino acid changes are preserved, which are unique in different denv- genotypes (table ) . these substitutions resulted in changes of its polarity, hydrophobicity or charge. especially notable was the change from l to s at position of the ns region, which is an amino acid substitution unique to genotype ii. this might be the result of positive selection within the lineage of genotype ii but not other genotypes. all denv- isolates from thailand belong to genotype ii, and interestingly, based on a previous publication [ ] , strains of denv- isolated prior to in thailand may have been replaced by two new locally evolving strains. this could be a sign of a new genotype evolving in thailand; however, most of the mutations or substitutions occurring were deleterious and a purifying selection of denv- was suggested [ ] . it is very likely that the previous analysis focused on only the e protein gene. determining the possibility of a positive natural selection site in the non-structural genes of the new thailand lineage will require further study. a number of t-and b-cell epitopes are present on the non-structural proteins, especially the ns gene [ ] [ ] [ ] . even though the biological significance of the l to s change at position of the ns region is unclear, growing evidence supported by in vitro and in vivo studies suggest that there are certain evolutionary forces acting on the ns gene shaping the gene flow of the dengue viral population, which might differ during viral replication in mammalian and mosquito cells [ , ] . this is the first time that a positive selection pressure site was detected in a non-structural protein in denv- and its importance together with its functional relevance to epidemic severity will need to be examined with a larger sample size. the global distribution of different genotypes of denv- indicates that they originated in southeast asia; these genotypes demonstrated higher epidemic potential with regards to severe dhf epidemics in sri lanka, central and south america [ , ] . genotype iii, once its transmission cycle was established locally, soon resulted in dhf epidemics regardless of an increase in virus transmission or a change in circulating serotypes [ , ] , supporting the hypothesis that virus strain is an important risk factor for dhf [ , ] . two sub-lineages (isolated before and after ) existed within the denv- genotype iii strains from sri lanka, and the viruses isolated after were associated with the dhf epidemic [ ] . we found that the strain isolated in from a indigenous dengue patient ( tw ) that did not lead to a large-scale epidemic of df or dhf was more closely related to the lineage of the denv- genotype iii sri lankan strain isolated before . similarly, in indonesia two sub-lineages of denv- were present (isolated before and after ), and a greater dhf epidemic, especially in adult cases, was caused by the denv- strains isolated after [ ] . the denv- strain isolated in taiwan during the dhf outbreak in was actually more closely related to the old indonesian strain of genotype i from - . while it is currently unknown how the different sub-lineages within each genotype are associated with different dhf epidemic potential, a recent publication suggested that changing serotype prevalence could lead to differential susceptibility to cross-reactive immune responses [ ] . furthermore, wearing et al suggested that both vector and short-termed host cross-immunity are two factors responsible for dengue epidemics [ ] . it would be necessary to strengthen comprehensive dengue virological surveillance, especially in those endemic and hyper-endemic areas/countries, to monitor the emergence of denv strains with epidemic potential for better epidemic prevention and vaccine 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dengue virus type nonstructural proteins ns a, ns b, and ns inhibition of interferon signaling by dengue virus inhibition of alpha/beta interferon signaling by the ns b protein of flaviviruses precise location of sequential dengue virus subcomplex and complex b cell epitopes on the nonstructural- glycoprotein recognition of snthetic oligopeptides from nonstructural proteins ns and ns of dengue- virus by sera from dengue virus-infected children development and evaluation of serotype-and group-specific fluorogenic reverse transcriptase pcr (taqman) assays for dengue virus temperature sensitive mutations in the genes encoding the ns , ns a, ns , and ns nonstructural proteins of dengue virus type restrict replication in the brains of mice e/ns modifications of dengue virus after serial passages in mammalian and/or mosquito cells phylogenetic analysis of dengue- viruses prevalent in guatemala during - internaltional notes dengue type infection -nicaragua and panama cities spawn epidemic dengue viruses disease exacerbation caused by sequential dengue infections: myth or reality? evolution of the dengue- virus in indonesia and throughout se asia may contribute to recent changes in outbreak dynamics ecological and immunological determinants of dengue epidemics we sincerely thank shih-ting ho at the sin-lau christian hospital, chien-ming li at the chi-mei foundation medical center and shih-chung lin at the kuo general hospital for cooperation in kindly providing the clinical samples. the study was supported by the grants from the national health research institute (nhri), taipei, taiwan (grant number: nhri#dd - x-cr- p and nhri#cn-cl p) and the national science council (nsc# - -b- - , nsc# - -b- - ) in taiwan. the authors declare that they have no competing interests. dyc and cck designed and performed all the experiments and drafted this manuscript together. dyc participated in the sequence alignment and statistical analysis. jhh and ycw helped with collecting field human isolates and ljc helped with sequencing experiments, together. thl helped for the field mosquito collection and gjc for- key: cord- -u y ttw authors: chen, keyan; zhao, kui; song, deguang; he, wenqi; gao, wei; zhao, chuanbo; wang, chengli; gao, feng title: development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: u y ttw background: the incidence of phe among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. therefore, the development of a sensitive, specific, and easily-performed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. results: an immunochromatographic strip was developed for the detection of phe-cov. the colloidal gold-labeled mab d was used as the detection reagent, and the mab e and goat anti-mouse igg coated the strip's test and control lines, respectively. the immunochromatographic strip was capable of specifically detecting phe-cov with a ha unit of within min. storage of the strips at room temperature for months or at °c for months did not change their sensitivity or specificity. using rt-pcr as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be % and . %, respectively. there was an excellent agreement between the results obtained by rt-pcr and the immunochromatographic strips (kappa = . ). additionally, there was a strong agreement between the sandwich enzyme-linked immunosorbent assay (elisa) and immunochromatographic strips (kappa = . ). when the immunochromatographic strips were used for diagnosing phe-cov infection in the jilin province, the phe-cov-positive rate ranged from . % in the jilin district to . % in the songyuan district. conclusions: based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of phe-cov for surveillance and epidemiological purposes. porcine hemagglutinating encephalomyelitis (phe) is an infectious disease primarily affecting pigs under weeks of age, causing vomiting, exhaustion, and obvious neurological symptoms [ ] . the mortality rate varies between - % [ ] . the disease is caused by phe coronavirus (phe-cov), which comprises a single strain and is the only known neurotropic cov affecting pigs [ ] . phe-cov was isolated for the first time in vivo from breastfeeding pigs suffering from encephalomyelitis in canada [ ] . in , an antigenically identical virus was isolated in england from suckling pigs presenting with anorexia, depression, and vomiting, but without clear signs of encephalomyelitis [ ] . surviving animals remained stunted in growth, and the condition was therefore called 'vomiting and wasting disease' (vwd). in china, phe-cov was first reported in , and it was later described both on the mainland and in the taiwan province [ , ] . the infection has also been reported in the major pig raising countries of europe, asia and north america, where it seemed to be endemic with no clinical outbreaks [ ] [ ] [ ] . currently, the incidence of phe among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. in , phe-cov was isolated from newborn and early-weaned pigs with vomiting and posterior paralysis on a canadian farm [ ] . in , this disease occurred twice in pig farms in the jilin province, with incidence rates among -dayold piglets as high as % and reported mortality rates ranging from % and % [ ] . in august , some pig farms in argentina experienced outbreaks of this disease, leading to deaths, morbidity rates as high as . % and a mortality rate of . % [ ] . therefore, early detection and control of phe-cov infection would be significant both from an economic and health viewpoint. at present, various laboratory methods are available for the detection and surveillance of phe-cov, including virus isolation [ ] , hemagglutination/hemagglutination inhibition (ha/hi) tests [ ] , immunohistochemistry (ihc) assays [ ] , and molecular tools such as nestedpolymerase chain reaction (nested pcr) and reverse transcriptase-polymerase chain reaction (rt-pcr) that enable detection of specific cov rna sequences from infected tissues [ , ] . however, these detection methods are laborious, time-consuming, and require laboratory procedures or special equipment, making them unsuitable for on-site inspection. current detection strategies are also insufficient to meet the needs of emergent management after phe outbreaks, thus restricting their application to veterinary clinical diagnosis. therefore, the development of a sensitive, specific, and easilyperformed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. an immunochromatographic assay is a unique immunoassay developed in in which a cellulose membrane is used as the carrier and a colloidal gold-labeled antigen or antibody is used as the tracer [ ] . it combines the immune response with chromatographic theory in a test that is simple and quick, providing specific, sensitive, and clear results with simple or no instrumentation. thus, it is suitable for testing clinical samples on site, in clinics and in locales where medical treatment and laboratory facilities are not available. an immunochromatographic assay has been widely used for animal quarantine and medical reasons [ , ] . ( ) ( ) ( ) in this study, an immunochromatographic strip with high sensitivity and specificity was developed for the detection of phe-cov, combining monoclonal antibody (mab) and colloidal gold immunochromatography (gica), and the resulting product is suitable for the surveillance of phe-cov. balb/c mice were purchased from the laboratory animal center of general hospital of shenyang military area command in china. animal immunization experiments were performed in accordance with the guidelines for animal experimentation of the general hospital of shenyang military area command. the animals were maintained under pathogen-free conditions. the field samples (brain tissue samples from deceased piglets and nasal cavity or throat swabs from ill piglets) were provided by jilin center for disease control and prevention in order to perform general surveillance on the phe-cov infection. the virus strain used in this study was phe-cov- n (genbank accession no. ay ). the viruses were propagated and passaged in porcine kidney epithelial (pk)- cells [ ] , and purified by sucrose density gradient centrifugation. the viruses were stored at − °c until needed. the procedure employed for the production of the monoclonal antibodies (mab) against the he and s proteins of phe-cov were based on the protocol of kohler and milstein [ ] ,and the recombinant s protein of phe-cov were produced as previously described [ ] . briefly, balb/c mice at weeks of age were immunized subcutaneously with . ml ( units of ha) of phe-cov virus purified by sucrose density gradient centrifugation and emulsified : with freund's complete adjuvant (sigma, st. louis, mo). the mice were boosted three times with the same amount of antigen in % freund's incomplete adjuvant (sigma, st. louis, mo) every weeks, followed by an intraperitoneal injection of . ml phe-cov. three days later, their splenic mononuclear cells were isolated and fused with murine myeloma cells (sp / ) using % polyethylene glycol (peg)- (sigma, st. louis, mo). the hybridomas were generated through the selection of hat (sigma, st. louis, mo) medium and screened using a recombinant s protein-based enzyme-linked immunosorbent assay (elisa) and hi assays. the positive hybridoma cells were cloned by a limiting dilution to obtain four strains of secretory positive antibody hybridoma cells. the stable hybridoma clones of the immunoglobulin g mab d and e were injected into balb/c nude mice and the mouse abdominal dropsy was purified by sequential precipitation with caprylic acid [ ] and ammonium sulfate and dialyzed against phosphate buffer ( . m, ph . ) at °c. their purities were used in western blot analysis. the sandwich enzyme-linked immunosorbent assay (elisa) the elisa assay was based on the mab of anti-phe-cov for the detection of phe-cov. the purified mab d of phe-cov, diluted to a final concentration of . μg/ml with carbonate buffer ( . m, ph . ), was used as the capture antibody in elisa for coating well microtiter plates (costar corning inc., corning, ny.) with μl per well. the plate was incubated overnight at °c. the plates were washed times with pbs-tween (pbst), and nonspecific binding sites were blocked with % (w/v) bovine serum albumin (bsa) (sigma, st. louis, mo) in pbst ( μl/well) for h at °c. after washing the plates, the samples of the homogenate grind suspension or the supernatant of the cell cultures were diluted : with pbst containing % (w/v) bsa, and μl was added into the coated well. the phe-cov and pbst standards were added as positive and negative controls, respectively, and the samples were incubated for . h at °c. after washing the plates, the mab e ( μg/ml) was added to each well at μl per well, the samples were incubated for . h at °c, and the plates were washed times with pbst. horseradish peroxidase-conjugated goat anti-mouse igg (sigma, st. louis, mo) was added into each well at a working concentration of : , incubated for h at °c, and the plates were washed times with pbst. one hundred microliters of substrate solution o-phenylenediamine (opd containing h o ) was added to each well, and the color reaction was developed in the dark for minutes at room temperature. the reaction was then stopped with m of h so μl/hole, and the absorbance was read at od with a universal microplate reader (bio-rad laboratories inc., richmond, ca). colloidal gold was prepared as previously reported [ ] , with minor modifications. briefly, ml of . % (wt/ vol) haucl in doubly distilled water in a -ml siliconized conical flask was heated to boiling in a microwave oven, and then . ml % trisodium citrate was added to the solution. after the colloidal gold solution was boiled for an additional min, it turned a cardinal red color and was allowed to cool gradually and was stored at °c in a dark-colored glass bottle. the ph of the colloidal gold was adjusted to . with % potassium carbonate (wt/vol), followed by sub-installing tubes with ml colloidal gold solution each, to which were added μg, μg, μg, μg, μg, μg, μg, μg, or μg mab d . tubes were shaken for min, and then . ml of % nacl solution was added to each tube and mixed. two hours later, results were recorded. the mab d antibody ( . μl, . mg/ml) was added dropwise into ml of colloidal gold solution on a magnetic stirring apparatus for min, stood at °c for min, and then ml % (wt/vol) bsa was added to block excess reactivity of the gold colloid. the mixture was then stirred on the magnetic stirring apparatus for an additional min and stored at °c for h. after the mixture was centrifuged at , × g at °c for min, the supernatant was centrifuged at , × g at °c for min, and the resulting conjugate pellet was suspended in mm borax buffer (ph . ) containing % (wt/vol) bsa and . % nan . the sizes and shapes of the unconjugated colloidal gold and colloidal gold conjugated to antibodies were characterized using transmission electron microscopy. the immunochromatographic test device consisted of a plastic support to which an immunochromatographic strip composed of a sample pad, a conjugate pad, a nitrocellulose (nc) membrane, and an absorbent pad were mounted. the colloidal gold-labeled mab d solution was dispensed onto glass fiber paper at a speed of μl per cm using an xyz dispense workstation (beckman, usa), and the conjugate pad was dried under vacuum. the mab e ( . mg/ml) or the goat anti-mouse antibody ( mg/ml) was dispensed at the test or the control line on the nc membrane, at a rate of . μl/cm and a speed of cm/s using xyz- , and the membrane was dried under vacuum and stored at °c. the sample pad, pretreated conjugate pad, nc membrane, and absorbent pads were glued together on a support board and assembled into a test strip plate. then the strip plate was cut into -mm-wide pieces using an ln- cutting machine (beckman, usa). in addition, a sample pad completed the assembly with . -to . -mm overlap sequentially by mounting on the conjugate plastic card (figure ). the strips were stored in dry conditions at °c until required. during testing, approximately μl supernatant of the antigen samples were added to the sample hole of the immunochromatographic strip, and this liquid rapidly diffused into the conjugate pad. for positive samples, phe-cov was captured by mab d through percolation in the nitrocellulose membrane, while in the test line, the formation of a colloidal gold mab d -phe-cov-mab e complex caused the appearance of a red line; for negative samples, the test line zone did not form a colloidal gold mab d -phe-cov-mab e complex, and therefore no red line was evident. as a control, both the negative and positive sample control lines turned red by forming a colloidal gold mab d -goat anti-mouse igg complex; otherwise, the test results were invalid. the immunochromatographic strip is illustrated in figure . to evaluate the specificity of the immunochromatographic strips, phe-cov and other viruses, including tgev, pedv, prv, hcv, bcv, mhv and hcv-oc were simultaneously tested using the test strips. the virus samples of phe-cov ( units of ha) was diluted to : , : , : , : , : , : , : , : , and : with mmol/l borate buffer solution (ph . ), and these samples were simultaneously tested using the immunochromatographic strips to evaluate the strips' sensitivity. the same procedure was repeated three times by different operators. to determine the reproducibility of the strip, the same batch and five different batches of the immunochromatographic strip were used to detect phe-cov. all samples were repeated times and the data were collected. the immunochromatographic strips were stored at room temperature or at °c and used for testing positive ( ha units) and negative samples every months, to determine the stability of the test. to evaluate the correlation between the immunochromatographic strip and reference methods, a total of brain tissue samples were collected from deceased piglets with suspected phe-cov infection from several pig farms in the jilin province. a mg of brain tissue was measured and homogenized, then suspended : with pbs, followed by centrifuging at × g for min. the supernatant was collected for testing by elisa, the immunochromatographic strips, rt-pcr. the detection assay of phe-cov by rt-pcr were established as previously described [ ] . the kappa statistic [ ] and < . represented excellent agreement, good to fair agreement, and poor agreement, respectively [ ] . the immunochromatographic strips were applied to the diagnosis of phe-cov infection in the field. a total of nasal cavity or throat swabs were collected from approximately -to -week-old piglets, with vomiting and neurological symptoms consistent with phe-cov infection, from herds in the changchun, jilin, songyuan, siping, baishan, and liaoyuan districts of the jilin province, china in . the mab of anti-phe-cov were used in western blot analysis to identify the d mab, which recognizes the he protein, and the e mab, which recognizes s protein ( figure ) and was stored at − °c until use. the sandwich enzyme-linked immunosorbent assay (elisa) thirty negative and seven positive virus samples were detected by elisa (figure -a) . the threshold value of . was identified using a roc curve (figure -b ). an od > . indicated a positive result and ≤ . a negative result. additionally, phe-cov was diluted to ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, ng/ml, . ng/ml, . ng/ml, and . ng/ml with pbs, which was detected by elisa. the standard curve was drawn by curveexpert software with phe-cov concentration on the x-axis and the average od value on the y-axis (figure -c) . the linear regression constant r was . , and the linear detection range was . - ng/ml. good linearity was observed, as the lowest detection limit was . ng/ml. to determine the optimal concentration of monoclonal antibody with colloidal gold, different concentrations of d mab were added in tubes with ml colloidal gold solution each. as shown in figure . the color of the tubes without sufficient protein changed from red to blue, whereas the color of the tubes remained unchanged if the amount of protein exceeded the minimum needed. the amount of protein in the lowest colloidal gold concentration tube, which remained red, was increased by % when the optimal antibody concentration was reached. the appearance of the colloidal gold solution was deep red and translucent, with a bright color. a bright band was visible when the test card faced the sun. the colloidal gold particles were consistent in size and uniformly distributed, with a mean diameter of about nm (figure -a) when observed under a transmission electron microscope. the colloidal gold-labeled mab d was observed with the transmission electron microscope to be evenly distributed, and the particle size was consistent. the colloidal gold particles had a visible clear space around the halo, then the surface of proteins and other adsorbed particles (figure -b). to determine the specificity of the immunochromatographic strip, phe-cov was simultaneously tested with tgev, hcv, pedv, prv, bcv, mhv and hcv-oc using the immunochromatographic strips. clearly, while each of the other samples resulted in one strong band on the control line, phe-cov displayed an additional band on the test line of the immunochromatographic strips ( figure ) . phe-cov was detected at different dilution strengths and repeated in triplicate with the immunochromatographic strip. the results are shown in figure . when the antigen were diluted from : to : (from to ha units, respectively), the reaction on the test and control lines were observed. thus, the sensitivity of the phe-cov colloidal gold immunochromatographic test was determined to be units of ha antigens. this procedure was repeated five times to detect the phe-cov of ha units at by the same batch and then by five different batches to determine reproducibility. these results (table ) showed little variability within the same batch or in different batches, demonstrating that the detection of the virus is highly reproducible. in addition, the stability of the immunochromatographic strip under various storage conditions was determined. the results are shown in table , they were stored at room temperature for months with a reduction in sensitivity of % and at °c for more than months with no loss of sensitivity or specificity for the detection of phe-cov. to evaluate the agreement between the immunochromatographic strip and reference methods, the clinical samples from deceased piglets were detected by sandwich elisa, rt-pcr, and the immunochromatographic figure the virus samples were tested by enzyme-linked immunosorbent assay (elisa). sample numbers - were negative and - were positive. a, a histogram of elisa results from both positive and negative virus samples; b, spss . for windows was used to create a roc curve to evaluate the threshold value; c, the standard curve was drawn by curveexpert software with the phe-cov concentration on the x-axis and the average od value on the y-axis. figure the optimal antibody concentration. the colloidal gold solution was made with different concentration of mab d . the color of the tubes without sufficient protein changed from red to blue, and when the amount of protein added to the tube exceeded the minimum needed, the color remained unchanged. table . of the clinical samples, were positive and were negative using the immunochromatographic strip, while were positive and were negative using elisa and rt-pcr. thus, the specificity and sensitivity of the immunochromatographic strip, as compared with elisa and rt-pcr, were % and . %, respectively. there were excellent agreement (kappa = . ) between the immunochromatographic strip to elisa and rt-pcr. the immunochromatographic strip was next applied to the diagnosis of phe-cov infection in the jilin province. characterization of these samples revealed that out of swab samples were positive for phe-cov infection (table ) . notably, the positive rate ranged from . % in the jilin district to . % in the songyuan district. these findings suggest that phe-cov is commonly transmitted in the jilin province, and the immunochromatographic strip can be used for the detection and differentiation of phe-cov in clinical diagnosis. phe-cov belongs to group of the coronaviridae family, a group characterized by the presence of a gene encoding the he protein [ ] . nucleotide sequence analysis of the region covering the s probe revealed . % nucleotide sequence homology to bcv and . % figure the colloidal gold and gold-labeled proteins were observed by electron microscopy. the results of transmission electron microscope imaging of colloidal gold and gold-labeled proteins. a: colloidal gold (× , ), b: gold-labeled mab d (× , ). homology to hcv-oc [ ] . although phe-cov causes two distinct clinical syndromes in pigs, only one serotype of the virus is known to exist. outbreaks of phe-cov-associated disease are now on the rise in many countries, inflicting considerable economic damage to the pig industry. remarkably, these recent isolates showed a high degree of genetic and antigenic homology with the reference strain phe-cov- n [ ] . currently, immunohistochemistry (ihc) for phe-cov, or molecular tools such as rt-pcr, enable the detection of specific cov rna sequences from infected tissues [ , ] . however, these detection techniques usually require special primers, a working laboratory, skilled technicians, and specialized equipment, making rapid and on-site detection of viruses in the field difficult. therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of phe-cov infection and transmission. colloidal gold immunochromatographic assay (gica) is convenient, is rapid, has high specificity and sensitivity, and can be performed either without instruments or with only a simple instrument, making it suitable for clinical diagnosis and drug testing purposes in almost any context [ ] [ ] [ ] . colloidal gold is a negatively charged hydrophobic rubber particle that maintains a stable colloidal system through electrostatic repulsion [ ] . the key to successful colloidal gold labeling lies in the preparation of a homogeneous mixture of monodispersed colloidal gold particles [ ] . the microwave oven method was chosen in this experiment because it provides even heating without manual shaking, creating colloidal gold particles that are consistent in size and uniformly distributed, with a mean diameter of about nm under transmission electron microscope. in the preparation of gold markers, proteins and gold glue particles should also be present in an appropriate ratio. if too many antibodies are added, the amount of free antibodies instead of gold-labeled antibodies will increase, which will result in a decreased titer of gold-labeled mab d , affecting the test's sensitivity. conversely, if too few antibodies are added, it is easy for the colloidal particles to aggregate and precipitate, especially during purification by ultracentrifugation. therefore, it is essential to add sufficient colloidal gold antibodies to maintain stability. the specificity and sensitivity of the immunochromatographic strip are largely dependent on the following factors. first, the quality of the mabs used in the strip test is crucial for the specificity and sensitivity of the strip. in this experiment, both of the mabs used recognize the he and s proteins of phe-cov and have a high affinity for their respective antigen epitopes. analysis of the specificity showed the strip to be specific for the detection of phe-cov, because it reacted with neither two coronaviruses (tgev and pedv) in pigs nor with other group viruses of the coronaviridae family (bcv, hcv-oc and mhv), the common virus hcv, or prv, to which the clinical symptoms of phe-cov are similar. in addition, careful selection of a membrane is critical for high specificity, sensitivity, and rapid detection as the wicking rate and speed of liquid diffusion on the membrane (millipore corp shf , a liner). the membrane used in this experiment was nitrocellulose membrane (millipore corp shf , a liner) with a membrane pore size of . - . μm, igg binding force constant of - μg/cm , and a chromatography rate of - s/cm. the membrane ensures antibody absorption and the leaching and binding of gold-labeled proteins. these advantages enable the test results to be sensitive and easy to read [ ] . phe-cov is able to replicate in the upper respiratory tract with or without producing clinical signs. phe-cov can be isolated from the nasal cavity, trachea, brain, and lungs of diseased or healthy pigs [ ] [ ] [ ] and the brain had the highest detection rate by nested pcr and rt-pcr [ ] . because of rt-pcr and elisa are also widely applied in clinical practice due to its high sensitivity and specificity [ , ] . thus, a lot of brain tissue samples were collected from deceased piglets with suspected phe-cov infection, and using rt-pcr and elisa as reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be % and . %, respectively. there was an excellent agreement among the immunochromatographic strip to elisa and rt-pcr (kappa = . ). the quantifying test result indicated the sensitivity of the strip test to be close to, or slightly less than, that of elisa and rt-pcr. however, the elisa and rt-pcr assay each step of the procedure can strongly influence the result if not performed well. the procedure must be strictly followed, with good quality control inside and outside the laboratory, and reagents that ensure the test quality must be selected. although elisa and rt-pcr was developed for the detection of phe-cov with high specific and sensitivity, it is not suitable for use outside of the research laboratory. in contrast, the procedure for the immunochromatographic strip described in this paper is less laborious and time-consuming than the elisa and rt-pcr method. taken together, the results suggested that an immunochromatographic strip was developed with high sensitivity and specificity for detecting phe-cov, which could be used for clinical applications. phe-cov is excreted for - days in oronasal secreions [ , ] , with transmission occurring through exposure to nasal secretions. thus, a total of nasal cavity or throat swabs were collected from approximately -to -week-old piglets, with vomiting and neurological symptoms consistent with phe-cov infection, and application of the immunochromatographic strips for diagnosis of phe-cov infection in the jilin province. the positive rate ranged from . % in the jilin district to . % in the songyuan district. notably, the phe-cov positive rate was . % in liaoyuan district in the current study. however, the serum antibody positive rate was only . % in the previous study [ ] . this may be associated with the samples collected from different herds. these findings suggest that phe-cov is commonly transmitted in these districts in the jilin province. notably, specific pathogen-free (spf) pigs, derived from germ-free pigs and given artificial milk, have been introduced extensively in many farms in this area [ , ] . because these animals lack antibodies to certain pathogenic agents, including phe-cov, their susceptibility to these agents may be greater. therefore, reliable testing methods are important for the diagnosis and prevention of phe-cov disease, which can cause fatal outbreaks in phe-cov seronegative farms. from the results of clinical signs, an immunochromatographic strip was developed for detecting phe-cov. the immunochromatographic strip is a simple and quick test that requires no special training to use, and the sensitivity and specificity of the strip were similar to the elisa and rt-pcr test. this test would be a valuable addition to clinical detection techniques for phe-cov, especially in areas where laboratory facilities are not available. hemagglutination/hemagglutination inhibition ihc: immunohistochemistry; nested pcr: nested-polymerase chain reaction reverse transcriptase-polymerase chain reaction; mab: monoclonal antibody; gica: colloidal gold immunochromatography enzyme-linked immunosorbent assay; bsa: bovine serum albumin; pbs: phosphate buffered saline; nc: nitrocellulose; tgev: transmissible gastro-enteritis virus; pedv: pig epidemic diarrhea virus; prv: pseudorabies virus; hcv: hog cholera virus bcv: bovine coronavirus; mhv: mouse hepatitis virus isolation and identification of hemagglutinating encephalomyelitis virus form pigs in taiwan sequence of ther ′-terminal end ( . kb) on the genome of porcine hemagglutinating encephalomyelitis virus: comparison with other hemagglutinating coronavirus serological comparison of hemagglutinating encephalomyelitis viruses isolated from different outbreaks a hemagglutinating virus producing encephalomyelitis in baby pigs vomiting and wasting disease of 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seroepizootiologic study of vomiting and wasting disease virus in pigs comparison of elisa for the detection of porcine serum antibodies to non-structural proteins of foot-and-mouth disease virus osterhaus ad: evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections characteristics of a coronavirus causing vomition and wasting in pigs. arch gesamte virus forsch development of an immunochromatographic strip for serological diagnosis of porcine hemagglutinating encephalomyelitis virus the produce technology of spf pig were application of modern raising pig in china submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank american journal experts for excellent grammar revisions of this paper. the authors are extremely grateful to the staff working in the veterinary stations in the jilin province for collecting porcine virus samples for this study. this study was supported by the national natural science the authors declare that they have no competing interests.authors' contribution kc and kz carried out most of the experiments and wrote the manuscript. ds and wh participated in the planning of the project. cz and gw collected the samples. fg and cw conceived of the study and participated in its design and coordination. all authors read and approved the final manuscript. key: cord- -f v wi h authors: ammayappan, arun; upadhyay, chitra; gelb, jack; vakharia, vikram n title: complete genomic sequence analysis of infectious bronchitis virus ark dpi strain and its evolution by recombination date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: f v wi h an infectious bronchitis virus arkansas dpi (ark dpi) virulent strain was sequenced, analyzed and compared with many different ibv strains and coronaviruses. the genome of ark dpi consists of , nucleotides, excluding poly (a) tail, and comprises ten open reading frames. comparative sequence analysis of ark dpi with other ibv strains shows striking similarity to the conn, gray, jmk, and ark , which were circulating during that time period. furthermore, comparison of the ark genome with other coronaviruses demonstrates a close relationship to turkey coronavirus. among non-structural genes, the 'untranslated region (utr), c-like proteinase ( cl(pro)) and the polymerase (rdrp) sequences are % identical to the gray strain. among structural genes, s has % identity with ark ; s has % identity with jmk and % to conn; b %, and c to n is % identical to conn strain. possible recombination sites were found at the intergenic region of spike gene, 'end of s and a gene. independent recombination events may have occurred in the entire genome of ark dpi, involving four different ibv strains, suggesting that genomic rna recombination may occur in any part of the genome at number of sites. hence, we speculate that the ark dpi strain originated from the conn strain, but diverged and evolved independently by point mutations and recombination between field strains. avian infectious bronchitis virus (ibv) is a pathogen of domestic chickens that causes acute, highly contagious respiratory disease [ ] . ibv is a member of the coronaviridae, order nidovirales [ ] and its genome consists of a . kb single stranded positive-sense rna molecule that encodes for four structural proteins; the spike (s) glycoprotein, the small envelope (e) protein, the membrane (m) glycoprotein, and the nucleocapsid (n) protein [ , ] . six subgenomic mrnas are transcribed from the ibv genome in virus-infected cells. the mrna contains two large overlapping open reading frames, encoding two polyproteins a and b [ ] , among which b is produced as ab polyprotein by ribosomal frame-shifting mechanism [ ] . many serotypes have been described for ibv, probably due to the frequent point mutations that occur in rna viruses and also due to recombination events demonstrated for ibv [ ] [ ] [ ] . for this reason, the characterization of virus isolates existing in the field is very important. the ark dpi strain was first isolated from delmarva peninsula broiler flock [ , ] and it is currently being used as a vaccine in the usa and europe. in this study, we characterized the entire genome of virulent ark dpi strain (embryo passage ) and compared it with other ibv strains and coronaviruses from all over the world. the ark dpi virus was inoculated into -day-old spf chicken eggs and allantoic fluid was collected h post inoculation. the fluid was clarified by low speed centrifugation and clear supernatant was stored at - °c. genomic rna was extracted from virus-infected allantoic fluid with qiagen rnaeasy kit, following the manufacturer's instructions, and stored at - °c until further use. oligonucleotides were designed based on consensus sequence of the following ibv strains: cal [gen-bank:ay ], mass [genbank:ay ] and bj [genbank:ay ]. overlapping primers were designed in a manner such that each pair of primer covered approximately two kb of genome. the rt-pcr was carried out as described earlier [ ] and the rt-pcr products were cloned into pcr . topo ta vector (invitrogen, ca). plasmid dna from various clones was sequenced by dideoxy chain termination method, using an automated dna sequencer (applied biosystems, ca). three independent clones were sequenced for each amplicon to exclude errors that can occur from rt and pcr reactions. the assembly of contiguous sequences and multiple sequence alignments were performed with the genedoc software [ ] . the pair-wise nucleotide identity and comparative sequence analyses were conducted using vector nti advance software (invitrogen, ca) and blast search, ncbi. phylogenetic analyses were conducted using the mega program [ ] . the genbank accession number for the ark dpi sequence is eu . the complete genomes of following strains are obtained from genbank: tcovmg , nc_ ; beaudette, nc_ ; m , ay ; ck/ch/lsd/ i, eu ; a , eu ; lx , ay ; saibk, dq ; the accession numbers of ibv gene sequences which are used in this study are as follows: for replicase gene sequences: the details of genome organization of ark dpi are shown in fig. . ibv polyprotein is cleaved into cleavage products, among which first two n-terminal products are cleaved by pl pro and rest of the c-terminal products are cleaved by cl pro [ ] . the putative domains and their cleavage sites ( fig. ) are predicted by comparison of amino acid sequences of each non-structural protein (nsp) of ark dpi with those of ibv-beaudette which is available in coronavirus database (covdb) [ ] . the nucleotide and the amino acid identity of ark dpi with other ibvs and coronaviruses are listed in tables , , . the whole structural gene of jilin is % identical to ark dpi, which suggests that jilin strain is actually ark dpi, which is currently used as a vaccine in china [ ] . the whole genome comparison of ibv strains reveals a close relationship of ark dpi with cal ( % identity), as shown in fig. . earlier studies have shown that cal probably evolved from ark dpi [ ] . the complete genome sequence analysis of ark dpi strain shows striking similarity to the conn, gray, jmk, and ark ibv strains, which were circulating during that time period [ , [ ] [ ] [ ] . the 'utr, pl pro , m pro and rdrp sequence analysis demonstrates that ark dpi is % identical to gray strain, except for pl pro which has % identity, as shown in table . it was suggested that pl pro gene has high genetic variation because of selection pressure [ ] . from this analysis, it appears that genetic mutation may have occurred at pl pro gene level. the modern strain ga maintains % identity with ark dpi in replicase proteins and because of unavailability of sequence information for rest of the genome; we speculate that ga may be a derivative of the ark dpi strain. analysis of the structural region of ark dpi clearly demonstrates that it is a chimera of three strains. the s gene of ark dpi is probably derived from ark ( % identical) and because of genetic mutations in the s region, ark dpi may have evolved independently. there is an a-t rich sequence tgtgttgattataat (fig. ) at the 'terminus of s gene (~ nts upstream from the end of s gene) which is conserved among most of the ibv strains. the s gene of ark maintains its identity with ark dpi up to this conserved region, but from this point onwards to the end of s , the nucleotide sequence is % identical to jmk strain. the recombination between jmk and ark had taken place presumably between above mentioned conserved region and intergenic (ig) region of s gene, which is located nts upstream of start codon of s gene. it is speculated that ig sequences serve as "hot spots" for recombination because of its consensus nature [ ] . gray and jmk strains share % homology both in the s and s genes of ark dpi, but jmk shows greater identity than gray strain, as shown in table . it is interesting that very few residues in the s gene make the gray strain nephrotropic, whereas jmk is pneumotropic [ ] . out of nts of gene a of ark dpi, last nts are % identical to conn, whereas first nts are only % identical. even though it is not clear whether the '-end of a was derived from conn or jmk, but it is evident that the recombination event may have occurred between jmk and conn at gene a. the b gene of ark dpi and conn differed only by two nucleotides and both share % identity, suggesting that b belongs to conn strain. from gene c to n gene, ark dpi shares % identity with conn. it is obvious that the entire structural genome, except spike, belongs to conn strain. cross protection studies carried out by gelb and coworkers [ ] demonstrated that the birds immunized with ark dpi showed classical genome organization of ibv-ark dpi %, % and % protection against conn, ark and jmk strains, respectively. indeed, these results suggest that major part of ark dpi genome was derived from conn. the level of protection for jmk is %, when ark was used as immunogen [ ] . on the other hand, conn and jmk immunization induces inadequate immunity against ark-type ibv challenge, suggesting that ark cross-immunity to jmk and conn is a one-way relationship [ , , ] . recombination hot spots have been demonstrated for ibv isolates by many researchers. these hot spots have been detected in the ig region [ ] , s gene [ ] , ' terminus of s , n and between n gene and 'utr [ , ] . some earlier sequencing studies had provided circumstantial evidence of recombination events in field isolates of ibv [ , , ] . more or less recombination sites were detected over the entire genome of coronavirus [ ] . based on these results, we speculate that the ark dpi strain originated from the conn strain, but diverged and evolved independently by point mutations and recombination between field strains. these findings suggest that there is high level of genetic diversity among currently circulating ibv serotypes. most of them come from genetic changes which already exist in the ibv field strains and from ibv live vaccines. so frequent monitoring is highly essential to track the emergence of new variants and is mandatory to develop efficient vaccination strategies to control and prevent ib. a sequences with > % identity are in bold letters b na-not analyzed c parental strains of ark dpi are shown in bold letters and immediate derivative of ark dpi is indicated by asterisk (*). phylogenetic tree analysis of complete ark dpi genome sequence with other ibv strains the propagation and cytopathogenic effect of an egg-adapted strain of infectious bronchitis virus in tissue culture infectious bronchitis coronaviruses: structure and genome expression cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus binns mm: completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot infectious bronchitis virus: evidence for recombination within the massachusetts serotype experimental evidence of recombination in coronavirus infectious bronchitis virus s gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel serologic and crossprotection studies with several infectious bronchitis virus isolates from delmarva-reared broiler chickens prevalence of arkansas-type infectious bronchitis virus in delmarva peninsula chickens molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus genedoc: analysis and visualization of genetic variation mega : molecular evolutionary genetics analysis (mega) software version . virus-encoded proteinases and proteolytic processing in the nidovirales covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes infectious bronchitis virus: s gene characteristics of vaccines used in china and efficacy of vaccination against heterologous strains from china genotypic and phenotypic characterization of the california (cal ) variant of infectious bronchitis virus immunological characteristics of a variant of infectious bronchitis virus isolated from chickens etiology of an infectious nephritisnephrosis syndrome of chickens a new serotype of infectious bronchitis virus responsible for respiratory disease in arkansas broiler flocks comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains evidence of genetic diversity generated by recombination among avian coronavirus ibv molecular cloning and sequence comparison of the s glycoprotein of the gray and jmk strains of avian infectious bronchitis virus cross-immunity in chickens using seven isolates of avian infectious bronchitis virus the neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures using dna shuffling to create novel infectious bronchitis virus s genes: implications for s gene recombination a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains evolution of avian coronavirus ibv: sequence of the matrix glycoprotein gene and intergenic region of several serotypes evidence of natural recombination within the s gene of infectious bronchitis virus. virology rna recombination in animal and plant viruses the authors declare that they have no competing interests. vnv and jg conceived the study. aa planned the experimental design, aa and cu carried out cloning and sequencing. aa drafted the manuscript. all authors critically reviewed and approved the final manuscript. key: cord- -sophqqne authors: chu, victor c; mcelroy, lisa j; aronson, jed m; oura, trisha j; harbison, carole e; bauman, beverley e; whittaker, gary r title: feline aminopeptidase n is not a functional receptor for avian infectious bronchitis virus date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: sophqqne background: coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (sars), and have the continued potential for emergence from animal species. a major factor in the host range of a coronavirus is its receptor utilization on host cells. in many cases, coronavirus-receptor interactions are well understood. however, a notable exception is the receptor utilization by group coronaviruses, including avian infectious bronchitis virus (ibv). feline aminopeptidase n (fapn) serves as a functional receptor for most group coronaviruses including feline infectious peritonitis virus (fipv), canine coronavirus, transmissible gastroenteritis virus (tgev), and human coronavirus e (hcov- e). a recent report has also suggested a role for fapn during ibv entry (miguel b, pharr gt, wang c: the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review. arch virol , : – . results: here we show that, whereas both transient transfection and constitutive expression of fapn on bhk- cells can rescue fipv and tgev infection in non-permissive bhk cells, fapn expression does not rescue infection by the prototype ibv strain mass . to account for the previous suggestion that fapn could serve as an ibv receptor, we show that feline cells can be infected with the prototype strain of ibv (mass ), but with low susceptibility compared to primary chick kidney cells. we also show that bhk- cells are slightly susceptible to certain ibv strains, including ark , ark_dpi, ca , and iowa (< . % efficiency), but this level of infection is not increased by fapn expression. conclusion: we conclude that fapn is not a functional receptor for ibv, the identity of which is currently under investigation. the family of coronaviridae is composed of group - coronaviruses (covs) [ ] . these viruses are able to infect human, canine, feline, murine, bovine, porcine, rat, and avian species. the etiological importance and zoonotic characteristics of coronaviruses have received much attention since the discovery of the newly emerged severe acute respiratory syndrome associated coronavirus (sars-cov) in [ , ] . coronaviruses have a high frequency of viral genome recombination and polymerase infidelity, which may have contributed to the increase of viral pathogenesis, inter-species transmission, and tissue tropism [ ] [ ] [ ] . in the case of sars-cov, its ancestral origin remains undetermined, but some evidence suggests that chinese horseshoe bats may be the natural reservoirs, while himalayan palm civets harbor and support inter-species transmission to humans [ , ] . other examples of extended tissue tropisms can also be found in some group covs. it is speculated that the acquisition of hemagglutinin esterase (he) activity from influenza c virus gives rise to the ability of sialic acid recognition and the extended tissue tropism and pathogenesis for some group covs [ ] [ ] [ ] . furthermore, bovine coronavirus (bcv) is thought to have jumped to human hosts, possibly by recombining with influenza c virus, thus giving rise to human coronavirus-oc (hcov-oc ) around [ , ] . receptor interaction between the virus and its host is the first step leading to a successful entry and productive replication. viruses increase fitness by adapting to environmental pressure through mutation and recombination. in contrast to other families of viruses that utilize a universal receptor to gain entry into host cells, members in the coronavirus family use a variety of cellular proteins and/or cofactors. group covs -including human coronavirus- e (hcov- e), feline infectious peritonitis virus (fipv), transmissible gastroenteritis virus (tgev) and canine coronavirus (ccv) -utilize human, feline, porcine, and canine aminopeptidase n (apn) as functional receptors during virus entry [ ] [ ] [ ] [ ] . the only notable exception is hcov-nl , which utilizes angiotensin-converting enzyme (ace ). in group cov, mouse hepatitis virus (mhv) of group a and sars-cov of group b independently utilize carcinoembryonic antigen-cell adhesion molecule (ceacam ) and ace to mediate infection [ , ] . however, other group a covs, including hcov-oc and bcov recognize n-acetyl- -oacetylneuraminic acid as a functional receptor [ ] . while the cellular receptors for both groups and covs have been identified and independently confirmed, group cov receptors remains undetermined. the avian covs, such as turkey cov and infectious bronchitis viruses (ibv), have been classified in group , with ibv the most extensively studied. recently, winter and colleagues suggested that sialic acids are responsible for ibv strain massachusetts entry [ ] . however, group covs lack he as a key viral protein regulating sialic acid binding, and the use of sialic acid would not explain the dependence on chicken cells for infection. therefore ibv is unlikely to use sialic acids as a functional entry receptor, but rather as a non-specific attachment factor. heparan sulfate may also serve as an attachment factor for the ibv strain beaudette (ibv_bdtt) [ ] . ibv_bdtt is a highly chicken embryo-adapted strain [ , ] , which has an extensive tropism in cell culture and efficiently infects various cell types, including bhk- cells [ , , ] . in contrast, clinical isolates and field strains of ibv typically only infect chicken cells. in the effort to identify the receptor for group covs, feline apn (fapn) was reported to allow entry of the ibv strain arkansas (ibv_ark ) [ ] . this could therefore be the first indication of a more universal receptor for the cov family. apn belongs to a family of metalloproteases [ ] . it is a type ii membrane-bound glycoprotein, and it is expressed on a variety of cell types, including granulocytes, monocytes, and fibroblasts. apn can also be found on the synaptic membranes of the central nervous system neurons, and on epithelial cells in the proximal convoluted tubules, intestinal brush border, and respiratory tract [ , ] . for coronaviruses in general, there is a crossspecies restriction that permits cells of a certain species to be infected only by its own complimentary cov. however, several studies on fipv and hcov- e, ccv and tgev have identified fapn as a universal entry receptor for group coronaviruses [ ] [ ] [ ] ] . the demonstration that fapn can allow infection by the ibv strain ark_ prompted us to examine both prototype and field isolates of ibv and test them for the potential use of fapn as a receptor. in this study, we first verified the use of the expressed fapn as a receptor for fipv and tgev by transient and constitutive expression of fapn in non-permissive bhk- cells. we also cultured seven strains of ibv, including arkansas , arkansas_dpi, california , connecticut , holland , iowa , and massachusetts (designated as ark , ark_dpi, ca , conn , h , iowa , and mass ) as candidates to test for fapn utilization by group avian covs. surprisingly, expression of fapn did not increase viral infection in any of the strains tested. as a consequence, we conclude that fapn is not a functional receptor during ibv entry. the authentic receptor is still under investigation. in order to determine fapn receptor usage by ibv, our goal was to rescue ibv infection in non-permissive cells by expressing fapn on the cell surface. to authenticate fapn expression, we first transiently transfected bhk- cells with fapn/pcdna . d/topo plasmid dna for hours. samples were then subjected to immunofluorescence staining to detect fapn protein expression (fig. a) . we found that nearly % of the bhk- population were efficiently transfected and stained positive for fapn expression. next, we verified the fapn expression on the bhkexp.fapn cell line that constitutively expresses fapn and its negative control cells, bhkexp.pcineo ( fig. b and c ). % of bhkexp.fapn cells also stained positive, while the negative control showed no fapn expression ( fig. ) . to verify the functionality of fapn as a coronavirus receptor, we first tested its ability to rescue fipv- and tgev infection of non-permissive cells, as reported in previous studies [ ] . feline kidney crfk cells and canine fibroblast a cells are able to support fipv- and tgev infection respectively (dr. edward dubovi, cornell university, personal communication). therefore, these cell lines served as positive controls for fipv- and tgev infection. we observed that fipv- and tgev efficiently infected crfk and a host cells respectively, but they were unable to mediate infection in bhk- cells ( fig. a and b), presumably due the lack of a functional receptor on the cell surface. however, transient transfec-tion of fapn in bhk- cells rescued fipv and tgev infection to % and % respectively (fig. c) . we therefore confirm that fapn can function as an entry receptor for both fipv- and tgev. group covs such as infectious bronchitis viruses (ibv) can infect primary chicken kidney cells at high efficiency, but they generally restrict interspecies infections [ ] . however, previous studies have shown that feline kidney cells (fek) can support ibv_ark infection [ ] , suggesting the presence of a feline receptor on the fek cell surface that enables ibv_ark entry. since ibv_mass has served as a prototype virus to study cov entry [ , ] , we first examined the potential utilization of a feline receptor by ibv_mass . we found that whereas bhk- cells were resistant to ibv_mass infection, and ibv_mass infected ck cells at % efficiency, ibv_mass was able to also infect feline crfk cells at % efficiency ( fig b) . as indicated by miguel et al. [ ] , the ibv_mass infection found in crfk cells suggests the potential use fapn as a receptor. to determine the potential role of fapn as a receptor for the prototype ibv (mass_ ), we transiently transfected fapn into bhk- cells. both untransfected and fapntransfected bhk- cells failed to show any detectable infection with ibv_mass , whereas primary ck cells were efficiently infected (fig. ) . to rule out the potential inhibitory effect of viral infectivity caused by transient transfection, we also examined ibv infection in cell lines constitutively expressing fapn. we found that expression of fapn in bhkexp.fapn cells could not rescue ibv_mass infection (fig. a and b ). to examine if fapn might act in a strain specific manner, seven field ibv isolates: ark , ark_dpi, ca , conn , h , iowa , and mass were obtained and cultured in -day old specific pathogen free (spf) chicken embryonic eggs. to verify virus infectivity, monolayers of ckc were individually incubated with all seven strains of ibv for h. figure shows that ckc monolayers were found to be efficiently infected by all seven field strains of ibv tested. to determine the role of fapn as a functional receptor for entry of these seven fields strains of ibv, as well as the prototype mass , were inoculated onto bhkexp.pcineo or bhkexp.fapn cell monolayers in triplicate. we found that bhk cells expressing empty vector alone (bhkexp.pcineo) were completely resistant to infection by ibv strains conn , h and mass . however, ibv strains ark , ark_dpi, ca , and iowa showed limited infection in the same cells, with a level of infection of less than . % (fig. a) . however, fapn expression did not significantly enhance ibv infectivity in any of the virus strains tested (fig. b) . consequently, our data show that ibv does not utilize fapn as a functional receptor during virus entry. viruses make use of a variety of receptors to gain entry into their target cells. the ability to recognize sialic acids that are ubiquitously present on the cell surface gives influenza viruses the ability to indiscriminately infect varied tissue or cell types. in contrast, retroviruses require more specialized receptor interactions between viral glycoprotein (gp ) and cd as well as other chemokine receptors on the t-helper lymphocytes during invasion [ ] . despite some subtle differences among different species or strains of viruses in the same family, they may still retain a trace of evolutionary similarity in terms of receptor utilization. however, receptor usage appears to have very little consensus among coronaviruses across different groups and species. coronaviruses seem to be able to devise various in order to gain a more comprehensive understanding of ibv receptor utilization, we cultured several clinical strains of ibv for this study. surprisingly, we found that the bhk- cells are in fact, weakly permissive to ibv_ark , ibv_dpi, ibv_ca , and ibv_iowa infections. however, fapn expression on bhk- cell surface did not increase viral infectivity for any ibv strain. we therefore conclude that fapn could not be a functional receptor for ibv entry. it is important to note that one, highly chick-embryo-adapted, ibv strain (ibv beaudette) gives efficient infection in bhk cells [ ] ; combined with the data presented here, that a low level of infection of bhk cells could be obtained with some clinical strains, this suggests that there are no post-entry restrictions to ibv replication and gene expression that might account for a lack of infection of bhk cells. how do we explain the discrepancy between these data and previous studies that show that fapn is the receptor for ibv_ark entry? according to our observations, ibv_ark viruses were able to infect bhk- cells to a limited degree (< . %), although the infection profile was not always consistent among samples. the relevance of this very limited infection of hamster cells is unclear. as a consequence, previous studies may have shown only localized and under-represented populations of cells, which were infected by ibv_ark , since there was no further quantification of viral infectivity. in contrast, our present study was performed in a quantitative manner by measuring infection frequency of nearly cells and we provide numerical virus infection data between fapnexpressing and non-expressing cells. various studies have shown several potential receptor candidates for group coronaviruses, which include fapn, sialic acids, and heparan sulfate [ , ] . however, there is still little consensus on which of these might be the functional entry receptor in vivo. sialic acid utilization as a functional entry receptor by ibv remains controversial since ibv generally has a highly restricted species tropism, and does not possess a he glycoprotein that would normally be responsible for sialic acid destruction and hence efficient virus spread. unlike influenza viruses that are established to use cell surface sialic acids for entry into a wide variety different cells, ibv is generally considered only to infect cells of chicken origin. the infected chickens often show lesions or lymphocyte infiltration in the ciliated epithelia cells, mucus secreting cells, and sub-epithelial cells along the respiratory tract [ ] . ibv also infects lung, ovary, and kidney tissues [ ] . in cell culture, ibv_mass infects ckc but not chick embryo fibroblast cells or the chicken df- fibroblast cell line (data not shown), suggesting possible cell type or tissue specificity for ibv infection. feline apn has been previously reported to serve as a functional receptor for ibv_ark entry, a conclusion based in part on the ability of the virus to infect feline kidney cells (crfk, also known as fek) [ ] . we have inde-ibv strain mass does not utilize fapn as an entry receptor pendently confirmed the ability of feline kidney cells to support ibv_mass infection (fig. a) with relatively high efficiency. as a consequence, cats could potentially serves as an intermediate host during an extension of coronavirus tissue tropism. in the case of sars-cov, viral transmission is thought to originate from chinese horseshoe bats to himalayan palm civet cats before jumping to humans [ , ] . the use of a feline receptor for ibv could potentially be another example of coronaviruses using feline cells as an interspecies reservoir to cross species boundaries. the reason why cats may serve as such an intermediate host is unclear, although it may be noteworthy that the endogenous feline coronavirus (fecv) can undergo distinct recombination events within feline cells that result in the emergence of the clinically different virus fipv [ ] . also of potential relevance is the suggestion that the sars-cov s gene may be a mosaic of feline and avian sequences [ ] . we conclude here that ibv cannot utilize fapn as a functional receptor. it is however important to note that although ibv does not utilize fapn as an entry receptor, there is still a possibility that chicken apn (capn) may serve as the key receptor. future work will address the potential usage of capn, as well as other chicken cell-specific proteins, as ibv receptors. ibv conn , iowa , h , and mass were obtained from dr. benjamin lucio-martinez, unit of avian health, cornell university, and ibv ark_dpi was obtained from dr. mark w. jackwood, university of georgia. ibv ark and ca were obtained dr. shankar p. mondal (university of california, davis). viruses were propagated in day-old embryonated chicken eggs and purified as described previously [ ] . virus stocks were titered by infection of primary chicken kidney cells, followed by immunofluorescence microcopy with monoclonal anti-s antibodies after - h of infection. typically, the highest titer stocks were able to infect between % and % of the ck cells present, and were used at this concentration for all infections, unless otherwise indicated. fipv- and tgev were obtained from dr. edward j. dubovi, animal health diagnostic center, cornell university, and were propagated in crfk (feline kidney cell) and a (canine fibroblast cells) respectively. bhk- cells were purchased from atcc and propagated twice weekly in dmem, supplemented with % fetal bovine serum (fbs) % penicillin-streptomycin, and mm hepes (cellgro). crfk and a were grown in l:m media. bhkexp.fapn or and bhkexp.pcineo cells were kindly provided by dr. kathryn v. holmes (university of ibv field isolates efficiently infect primary chicken kidney cells figure ibv field isolates efficiently infect primary chicken kidney cells. monolayers of ck cells were separately infected with ibv strain ark , ark_dpi, ca , conn , h , iowa , and mass for h. cell monolayers were labeled with monoclonal α-s or m ( : , : , or : ) antibodies to detect viral infection. fapn is not a functional receptor for field strains of ibv figure fapn is not a functional receptor for field strains of ibv. bhkexp.pcineo (a) or bhkexp.fapn (b) cells were independently infected with ibv strain ark , ark_dpi, ca , iowa , conn , h , and mass for h. cell monolayers were labeled with monoclonal α-s or m ( : , : , or : ) antibodies to detect and determine viral infections. viral infectivity was determined by counting approximately cells in three separate experiments. primary chicken kidney cells (ckc) were prepared as follows: in short, spf white leghorn chicks ( - days) were sacrificed in a co chamber, and kidneys were removed via aseptic techniques. kidney cells were washed in ml sterile phosphate buffer saline (pbs) twice via gentle stirring to remove red blood cells. the cells were then treated with ml of trypsin-edta (cellgro) for two minutes to separate large chunks of kidney tissues. cell supernatant was mixed with ml of fbs and centrifuged at , rpm for min. media were removed and kidney cells were resuspended in ml of m media. cell population was determined on a hemocytometer. for plating, - . × cell/ml was prepared in m media (m media with % fbs) and incubated in % co at °c. for infection, % confluent cell monolayers were inoculated with virus stocks and incubated for , , or hours before fixation. fapn/pcdna . d/topo and hapn/pcineo plasmid dnas were provided by dr. kathryn v. holmes (university of colorado health sciences). for standard transfection, . µg of plasmid dna were premixed with µl of lipofectamine (invitrogen) in µl of opti-mem (gibco) at room temperature according to manufacture's instructions. cell monolayers were transfected at °c for hours before fixation or virus infection. infection and immunofluorescence microscopy were essentially performed as described previously [ ] except with methanol fixation for detecting viral antigens. for fapn staining, cell monolayers were fixed in % paraformaldehyde at room temperature and labeled with monoclonal antibody r-g- (provided by dr. tsutomu hohdatsu, kitasato university, towada, aomori, japan). for viral antigen staining, anti-s monoclonal antibody : was used for ibv massachusetts and iowa . the anti-s monoclonal antibody : was used for ibv arkansas_dpi. the anti-m monoclonal antibody : was used for ibv arkansas , connecticut , california , and holland . monoclonal antibody b and rabbit polyclonal antibody were provided by dr. edward dubovi, cornell university, and were used detect fipv and tgev infection respectively. the secondary antibodies alex-fluor goat anti-mouse igg and alex-fluor goat anti-rabbit igg were purchased from molecular probes. cell nuclei were counter-stained with hoechst (molecular probes). cells were viewed on a nikon eclipse e fluorescence microscope. images were captured with a spot rt monochrome camera and spot advanced v. . . software and processed with adobe photoshop v. . virus taxonomy: family coronaviridae wet markets--a continuing source of severe acute respiratory syndrome and influenza? lancet high-frequency rna recombination of murine coronaviruses complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event animal origins of the severe acute respiratory syndrome coronavirus: insight from ace -s-protein interactions severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats sequence of mouse hepatitis virus a mrna : indications for rna recombination between coronaviruses and influenza c virus the hemagglutinin/esterase gene of human coronavirus strain oc : phylogenetic relationships to bovine and murine coronaviruses and influenza c virus sialic acids as receptor determinants for coronaviruses human aminopeptidase n is a receptor for human coronavirus e feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n is a receptor for all group i coronaviruses spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection angiotensin-converting enzyme is a functional receptor for the sars coronavirus sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus heparan sulfate is a selective attachment factor for the avian coronavirus ibv beaudette. avian diseases cultivation of the virus of infectious bronchitis new jersey agricultural experiment station studies on avian infectious bronchitis virus (ibv). ii. propagation of ibv in several cultured cells the avian coronavirus infectious bronchitis virus undergoes direct low-ph-dependent fusion activation during entry into host cells the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review families of zinc metalloproteases identification of residues critical for the human coronavirus e receptor function of human aminopeptidase n infectious bronchitis fluorescence dequenching assays of coronavirus fusion retrovirus receptors feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses mosaic evolution of the severe acute respiratory syndrome coronavirus influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis we thank (in alphabetical order) dr. joel d. baines, dr. edward j. dubovi, dr. tsutomu hohdatsu, dr. kathryn v. holmes, dr. mark w. jackwood, dr. benjamin lucio-martinez, and dr. shankar p. mondal for their generous support on providing various reagents possible for this study. we thank a. damon ferguson for her skillful technical assistance, and all members of the whittaker laboratory for helpful suggestions. this work was supported by grant r ai from the national institutes of health. the author(s) declare that they have no competing interests. vc participated in the design of the study, carried out the experiments presented, and drafted the manuscript. lm, jm, to and ch carried out preliminary experiments on which the manuscript is based. bb contributed to the conception and design of the experiments. gw conceived of the study, participated in its design and coordination, and wrote the final manuscript. all authors read and approved the final manuscript. key: cord- -lcuma eh authors: ashfaq, usman a; javed, tariq; rehman, sidra; nawaz, zafar; riazuddin, sheikh title: lysosomotropic agents as hcv entry inhibitors date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: lcuma eh hcv has two envelop proteins named as e and e which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. fusion of the hcv envelope proteins is triggered by low ph within the endosome. lysosomotropic agents (la) such as chloroquine and ammonium chloride (nh( )cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular ph. to investigate the antiviral effect of la (chloroquine and nh( )cl) on ph dependent endocytosis, hcv pseudoparticles (hcvpp) of a and a genotype were produced and used to infect liver cells. the toxicological effects of chloroquine and nh( )cl were tested in liver cells through mtt cell proliferation assay. for antiviral screening of chloroquine and nh( )cl, liver cells were infected with hcvpp of a and a genotype in the presence or absence of different concentrations of chloroquine and nh cl and there luciferase activity was determined by using a luminometer. the results demonstrated that chloroquine and nh( )cl showed more than % reduction of virus infectivity at μm and mm concentrations respectively. these results suggest that inhibition of hcv at fusion step by increasing the lysosomal ph will be better option to treat chronic hcv. hepatitis c virus (hcv) is an enveloped, positive stranded rna virus classified in the family of flaviviridae. hcv causes acute and chronic hepatitis infection which can eventually lead to permanent liver damage, hepatocellular carcinoma and death. it is estimated that three to four million people are infected with hcv every year [ ] . hcv genome encodes a single polyprotein precursor of just over amino acids. this polyprotein precursor is co-and posttranslationally processed by cellular (signal peptidase and signal peptide peptidase) and viral (ns - and ns - a) proteases to yield four structural (core, e , e and p ) and six non structural (ns , ns , ns a, ns b, ns a, ns b) proteins. hcv envelope is formed by e and e glycoprotein heterodimers which are essential for virus entry into cells [ ] . hcv e and e fusion was enhanced at low ph, suggesting that hcv enters cells via the endosomal pathway and that e and/or e undergo conformational modifications that allow fusion of viral and cellular membranes [ ] . there are six major and more than subtypes of hcv. this classification is based on nucleotide variation among different hcv isolates. they occur in different proportion in different parts of the world. genotype a and b are the most common genotypes in the united states and europe [ , ] . the most prevalent hcv genotype in pakistan is a followed by b and a [ ] . presently, there is no vaccine available for prevention of hcv infection due to high degree of strain variation. current therapeutic options for hepatitis c are limited, especially for genotype . for genotypes and , pegylated interferon in combination with ribavirin, can lead to a sustained virological response in up to % of patients [ ] . however, this therapy is expensive and often associated with side effects that may lead to discontinuation of therapy [ ] . hemolytic anemia, cough, shortness of breath & treatogenicity are the most common adverse effect associated with ribavirin treatment, and muscle aches, fatigue & neuropsychiatric adverse effects of ifnα lead to premature cessation of therapy in to % of patients [ , ] . moreover, cost of interferon for month treatment ranging from pkr , to , is beyond the financial range of most patients. hence, there is a need to develop anti hcv agents, which are less toxic, more efficacious and cost-effective. the term "lysosomotropic agent" was introduced by deduve and co-workers ( ) to designate substances that are taken up selectively into lysosomes. this definition leaves open the chemical nature of a lysosomotropic substance and the mechanism of its uptake. lysosomotrpic agents such as nh c , chloroquine and methylamine penetrate acidic compartments of the cell and accumulate as protonated forms, resulting in an increase in the intravesicular ph [ ] [ ] [ ] . chloroquine, which is widely used for the treatment of malaria, is a well-established inhibitor of autophagic proteolysis which acts by inhibiting acidification of lysosomes and endosomes [ ] . it has been reported that lysosomotropic agents such as chloroquine and nh cl exert direct antiviral effects on several rna viruses including coronaviruses, flaviviruses and human immunodeficiency virus (hiv) [ ] [ ] [ ] [ ] . moreover, clinical studies have demonstrated the safety, tolerability, and efficacy of lysosomotropic agents in the antiviral treatment of hiv infection [ , ] . in the current study hcv entry is blocked by lysosomotropic agents. firstly toxicological analysis of chloroquine and nh cl were done in liver cells. after toxicological analysis, antiviral effects were studied in hcvpp of a and a genotype. huh- and hek t cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal calf serum, iu/ml penicillin and μg/ ml streptomycin, at °c in an atmosphere of % co . huh- was kindly provided by dr. zafar nawaz (biochemistry and molecular biology department, university of miami, usa. the pcdna-e e expression vector encoding the e and e glycoproteins ( - ) of hcv genotype a and a, was generated by inserting into a nonpackageable, cmv promoter-driven expression construct. the cmv-gag-pol murine leukemia virus (mlv) packaging construct, encoding the mlv gag and pol genes, and the ptg-luciferase plasmid provided by dr jaean dubison, france. hcvpp were produced by co-transfection of -t cells with equal amounts of three expression vector as described previously [ ] . supernatants containing hcvpp were harvested h later, filtered through . μm pore-sized membranes and stored at - °c before use in infection of huh cells. mtt ( -[ , -dimethylthiazol- -yl]- , -diphenyltetrazolium bromide) is a rapid and sensitive in-vitro procedure for evaluating cellular toxicity of compounds. the mtt substance is reduced by mitochondrial succinic dehydrogenases in living cells to purple formazan crystals that are not soluble in aqueous water. the absorption of dissolved formazan in the visible region correlates with the number of viable cells [ ] . to investigate cellular toxicity, × huh- cells were plated into -well plates. after h, different concentrations of chloroquine and nh cl were added and the plate was sealed and kept at °c in an atmosphere of % co for h. after h, fresh media ( μl) and mtt solution ( mg/ml in pbs) were added to all wells in columns - . wrapped the plate in aluminium foil and incubated for - h at °c. media was carefully removed and added μl of dmso to dissolve the formazan crystals in columns - . mtt formazan product was determined by measuring absorbance with an enzyme-linked immunosorbent assay (elisa) plate reader at a test wavelength of nm and a reference wavelength of nm. cell viability was obtained using the following equation. percent cell viability = test nm − nm/control nm − nm × to investigate anti-fusion effect of chloroquine and nh cl, huh- cells were incubated in the presence or absence of chloroquine and nh cl at °c for min. after min huh- cells were infected with hcvpp of a and a genotype in the presence or absence of different concentrations of chloroquine and nh cl and incubated for additional h. after h cells were lysed and luciferase activity was determined by using a luminometer. all statistical analysis was done using spss software (version . , spss inc). data are presented as mean ± sd. numerical data were analyzed using student's t-test and anova. p value < . was considered statistically significant. mtt assay is a widely used test for evaluating cytotoxicity of compounds/herbs in cell cultures. the mtt substance is reduced by mitochondrial succinic dehydrogenases in living cells to purple formazan crystals that are not soluble in aqueous water. the absorption of dissolved formazan in the visible region correlates with the number of alive cells [ ] . cytotoxic effects of chloroquine and nh cl were analyzed after h incubation of huh- cells with different concentrations of compounds. figure showed that cell proliferation of liver cells is unaffected at high concentration. after toxicological analysis through mtt proliferation assay, antiviral activities of chloroquine and nh cl were tested at non toxic concentrations. enveloped viruses enter cells through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. fusion of the viral envelope protein(s) is triggered by low ph within the endosome. lysosomotropic agents such as chloroquine and nh cl, have been used to demonstrate the ph sensitivity of virus entry. we therefore tested the infectivity of hcvpp after treatment of target cells with different concentrations of chloroquine and nh cl. hcvpp of a and a genotype demonstrated dose-dependent inhibition in the presence of chloroquine and nh cl. chloroquine and nh cl showed greater than % reduction of virus infectivity at μm and mm concentration respectively, suggesting a ph-sensitive route of virus entry (figure a and b ). hcv entry is a multistep process requiring four cellular receptor, fusion and endocytosis. hcv fusion depends on e and e , viral dose, and occurs within a specific ph range. targeting ph dependent endocytosis is a useful tool to identify antiviral drugs against. a major advancement to look into hcv entry process was the development of hcvpp, consisting of native hcv envelope glycoproteins, e and e , assembled onto [ , , ] . this system is potentially very powerful tool to identify and characterize molecules that block hcv entry. in this study, hcvpp of local hcv genotype a and a were produced to study early entry steps mediated by hcv envelope glycoproteins. this assay is based on the quantification of retroviral dna synthesis, which occurs soon after the fusion of the retroviral particle with a cellular membrane. presumably, this assay is only dependent on the entry steps mediated by the heterodimer e e (binding, endocytosis, and fusion) and on the activity of the reverse transcriptase of the hcvpp retroviral core. the intracellular sub-compartments such as lysosomes and endosomes have an acid nature with ph of about [ ] . lysosomotropic agents such as chloroquine and nh cl are weak bases which have a tendency to accumulate in these compartments. lysosomotropic agents are captured by protonation inside the lysosomes and accumulate there (figure ). the ratio of intra/extra lysosomal concentrations of these substances is equal to the ratio of concentration of hydrogen ions in lysosomes and in their vicinity, i.e. : if we suppose that ph in lysosomes is and in cytoplasm . the amount of the permeable form of lysosomotropic agents passing through the membrane depends on the substance pka value and ph value of solution. the higher the pka value, the lower the permeable form ratio. this has led to the assumption that the specific uptake of lysosomotropic substances into lysosomes depends on the acidic ph of these compartments, ion-trapping weak bases [ ] . in this model, the most suitable weak bases are those with a pka around . nh cl and chloroquine are lysosomal weak bases that are known to affect acid vesicles leading to dysfunction of several proteins. previous studies of chloroquine and cl have demonstrated that it has multiple effects on mammalian cells in addition to the elevation of endosomal ph, including the prevention of terminal glycosylation of immunoglobulin's [ ] . when added to virusinfected cells, chloroquine inhibited later stages in vesicular stomatitis virus maturation by inhibiting the glycoprotein expression at the cell surface [ ] , and it inhibited the production of infectious hiv- particles by interfering with terminal glycosylation of the glycoprotein [ , ] . increase in ph by chloroquine and nh cl, firstly inhibits low ph confirmational changes that triggers fusion, penetration and uncoating and secondly inhibits posttranslational modification of hcv enveloped proteins (e and e ) (figure ). our results demonstrated that, chloroquine and nh cl inhibit hcvpp entry in a dose-dependent manner at non toxic concentration. chloroquine and nh cl resulted in greater than % reduction of virus infectivity at a concentration of μm and mm respectively, suggesting a ph-sensitive route of virus entry (figure a and b) . in conclusion, inhibition of hcv entry by increasing the ph through lysosomotropic agents is an important target to identified new drugs against hcv and combination of lysosomotropic agents with interferon will be better option to treat chronic hcv. hepatitis c virus infection the role of the hepatitis c virus glycoproteins in infection hepatitis c virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles geographical distribution of hepatitis c virus genotypes in blood donors: an international collaborative survey group atcs: hepatitis c virus type b (ii) infection in france and italy frequency distribution of hepatitis c virus genotypes in different geographical regions of pakistan and their possible routes of transmission chronic hepatitis c virus infection treatment of chronic hepatitis c with pegylated interferon and ribavirin ribavirin and interferon alfa- b in chronic hepatitis c: assessment of possible pharmacokinetic and pharmacodynamic interactions side effects of therapy for chronic hepatitis c weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes van hoof f: commentary. lysosomotropic agents effect of weak bases on the intralysosomal ph in mouse peritoneal macrophages chloroquine is a potent inhibitor of sars coronavirus infection and spread effects of chloroquine on viral infections: an old drug against today's diseases? anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity different ph requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza a viruses comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type hydroxychloroquine, hydroxyurea and didanosine as initial therapy for hiv-infected patients with low viral load: safety, efficacy and resistance profile after weeks rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays infectious hepatitis c virus pseudoparticles containing functional e -e envelope protein complexes cell surface expression of functional hepatitis c virus e and e glycoproteins chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine inhibition of human immunodeficiency virus infectivity by chloroquine anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors lysosomotropic agents as hcv entry inhibitors financial support by higher education commission pakistan is highly acknowledged. authors' contributions uaa, tj and sdr contributed equally in lab work and manuscript write up. zn and srd were the principal investigator and provide all facilities to complete this work. all the authors read and approved the final manuscript. usman ali ashfaq (phd molecular biology), imran shahid (m phil molecular biology), tariq javed (m. phil pharmaceutical chemistry, sidra rehman (msc chemistry) and sheikh riazuddin (phd molecular biology and dean post graduate study at allama iqbal medical college, lahore the authors declare that they have no competing interests. key: cord- - z qxz authors: harun, mohammad syamsul reza; kuan, choong oi; selvarajah, gayathri thevi; wei, tan sheau; arshad, siti suri; bejo, mohd hair; omar, abdul rahman title: transcriptional profiling of feline infectious peritonitis virus infection in crfk cells and in pbmcs from fip diagnosed cats date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: z qxz background: feline infectious peritonitis (fip) is a lethal systemic disease, caused by the fip virus (fipv); a virulent mutant of feline enteric coronavirus (fecv). currently, the viruses virulence determinants and host gene expressions during fipv infection are not fully understood. methods: rna sequencing of crandell rees feline kidney (crfk) cells, infected with fipv strain – at hours post infection (h.p.i), were sequenced using the illumina next generation sequencing approach. bioinformatic’s analysis, based on felis catus x annotated shotgun reference genome, using clc bio genome workbench mapped both control and infected cell reads to genes out of annotated genes. kal’s z test statistical analysis was used to analyse the differentially expressed genes from the infected crfk cells. real time rt-qpcr was developed for further transcriptional profiling of three genes (pd- , pd-l and a h) in infected crfk cells and peripheral blood mononuclear cells (pbmcs) from healthy and fip-diseased cats. results: based on kal’s z-test, with false discovery rate (fdr) < . and > . fold change on gene expressions, a total of genes were differentially expressed by both samples, where genes were up-regulated and the remainder were down-regulated. most genes were closely clustered together, suggesting a homogeneous expression. the majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and th cell functions, and the regulation of apoptosis. real time rt-qpcr developed focusing on up-regulated genes (pd-l and a h) together with an apoptosis associated gene pd- expressions in fipv infected crfk cells and in pbmcs from healthy and fip diagnosed cats produced concordant results with transcriptome data. conclusion: the possible roles of these genes, and their importance in feline coronaviruses infection, are discussed. feline coronaviruses are enveloped, positive sense rna viruses that can be classified into two biotypes, namely low virulent feline enteric coronavirus (fecv) and highly virulent feline infectious peritonitis virus (fipv). fecv is very common in the cat population worldwide, and has been shown to have infected - % pet cats and shed by - % cats in multi-cat environments [ , ] . of those shedding the virus, - % will develop feline infectious peritonitis (fip) disease [ ] . within the biotypes, the viruses are differentiated further into serotype i and serotype ii, based on virus neutralizing antibodies. type i fecv and fipv strains are more ubiquitous worldwide and are more likely to cause clinical fip, while type ii strains are less common, but more adaptable to cell culture [ ] . it has been suggested that fipv, the causative agent for fip, is a mutant form of fecv [ , ] ; where several possible nature of mutation responsible for the increase in virulence has been characterized. studies have shown that several mutations throughout the fipv genome were detected, but mutations at c membrane protein and b secretory glycoprotein genes were suggested to be responsible for transforming fecv to fipv [ , ] . a recent study revealed that mutation of the s /s locus and modulation of a furin recognition site normally present in the s gene of fecvs is a critical contributing factor for development of fip [ ] . furthermore, it was found that fipv infection is associated with t cell depletion by apoptosis; although the virus cannot infect cd + and cd + t cells [ , ] . therefore, apoptosis of cd + and cd + t cells is probably caused by mediators from infected macrophages and/or intestinal epithelial cells [ , ] . hence, little is known about the interaction of the virus and host cells; especially the early cellular transcriptional responses towards virus infection, virus mechanism of inducing t cell apoptosis, and the absence of cell-mediated immunity (cmi) response in fip infected cats. the use of a next generation sequencing approach in rna sequencing has facilitated understanding in defining the expression profiles of cellular responses during pathogen infections. this method has been proven to be helpful in explaining the pathogenesis of various viruses [ , ] , including feline immunodeficiency virus (fiv) infection [ , ] . furthermore, the availability of complete . x of cat genome, using the whole genome shotgun (wgs) approach, provides valuable information for the bioinformatic's analysis of feline host responses, following pathogen infection. moreover, the cat genome contigs were aligned, mapped, and annotated to ncbi annotated genome sequence of six index mammalian genomes (human, chimpanzee, mouse, rat, dog, and cow) using megablast [ ] . previous study has shown that more than % of fipv strain - were internalized by crfk cells at hours post infection [ ] . hence, in this study, mrna from crfk cells infected with fipv strain - at h.p.i were sequenced using illumina next generation sequencing technology. the generated data was then analyzed using clc bio genomic workbench, where the genes were compared to felis catus . x annotated shotgun reference genome. kal's z-test on expression proportions [ ] was used to determine significantly expressed genes. genes expressed with a false discovery rate (fdr) < . and > . fold change were considered for further analysis. overall, the trimmed sequence reads match to , annotated transcripts; where only ( . %) were statistically significant (kal's z test, p < . ), and out of the significant matched, only ( . %) transcripts were expressed with fold absolute change of or more. of these transcripts, ( . %) were up-regulated and the remainder were down-regulated. after blast analysis, up-regulated transcripts were matched to genes while down-regulated transcripts were matched to genes. of the up-regulated genes, there were transcripts per gene for genes, but only one transcript per gene for the remaining genes. meanwhile, of the down-regulated genes, there was one transcript per gene for genes and transcripts per gene for genes. as shown in figure , the rpkm of control samples was plotted against the rpkm of infected samples; and as such, genes with equal expression will line-up on the diagonal identity line while genes with different expression values will either be over or under the diagonal line. the further away the point is from the identity line, the larger is the difference between its expressions in one experiment compared with the other. except for genes (mx , rsad , plin , and serpinb ), most genes from both samples were closely clustered together, thus suggesting a homogeneous expression. the plot excluded genes (ccl , rnf , and rpl ) that had infinite fold change expressions. table shows the top up-regulated genes (in decreasing order) and their functions. the majority of the genes were those associated with immune response, while the remainder were associated with apoptosis, cell cycle, cytokine, and ubiquitination activities. furthermore, there were also interferon stimulated genes (isgs) coded for proteins (rsad , a c, a h, mda , ifi , and mx ) that were involved in inhibiting viral entry, replication, and production. interestingly, one gene (pd-l ), which negatively regulates immune response in viral infection, was also found to be highly up-regulated. meanwhile, the majority of the down-regulated genes were involved in pro-inflammatory cytokine's activation, cmi, and anti-apoptosis activities ( table ) . two unique downregulated genes (rnf and rpl ) were found to be expressed in control uninfected cells only, where the former had anti-apoptotic effect and the latter translated rna to protein. three host genes (a h, pd- , and pd-l ) were selected for real-time rt-qpcr analysis, because they were highly up-regulated and may play important roles during fipv induced disease; judging by their functions. a h was involved in viral rna and dna editing, causing mutation, while pd- and its ligand (pd-l ) were involved in programmed cell death that was associated with negative regulation of immune response. comparisons of fold change results of real-time pcr and transcriptome study for a h and pd-l genes revealed almost similar levels of fold changes. the transcriptome resulted in . and . fold change for transcripts of a h while real-time pcr resulted in . ± . fold changes (tables and ) . meanwhile, transcriptome resulted in . and . fold changes for transcripts of pd-l gene while real-time pcr resulted in . ± . fold changes. in the case of the pd- gene, rna sequencing was unable to detect the gene expression, due to low coverage (i.e., data not shown), although real-time pcr was able to detect an up-regulation of the gene at h.p.i. fipv induced high and exceptionally high a h expression at and h.p.i. respectively, but from to h.p.i., the gene was down-regulated. fipv infected cells also showed high up-regulation of pd- expression at h.p.i. and moderately up-regulated at h.p.i but were being down-regulated at , , and h.p.i. meanwhile, pd-l gene was consistently down-regulated from hours to h.p.i. peripheral blood mononuclear cells (pbmcs), obtained from cats with clinical signs associated with fip (table ) , were purified and analysed with real-time pcr. in general, all of the fip diagnosed cats expressed the pd- and pd-l genes more than folds, while only cats expressed a h gene more than folds ( table ). the highest expression fold for a h, pd- and pd-l genes was detected from cat no. at . ± . , . ± . , and . ± . fold changes, respectively. meanwhile, cat no. showed less fold changes of the genes compared to the other fip diagnosed cats. the pathogenesis of feline coronavirus infection is unclear. the reference feline genome sequence assembly of transcriptome analysis of early infection ( h.p.i.) of crfk cells with fipv - showed that the expressions of transcripts ( . % of the trimmed annotated) were statistically significant, based on kal's z test. only transcripts, which consisted of up-regulated genes and down-regulated genes, were expressed with fold absolute changes of or more. since only one sample per group was analysed, kal's z test was used to determine the significant differences in the expression profiles. study has shown that this test evaluates single sample against another single sample where each group in an experiment has only one sample [ ] . this test is based on an approximation of the binomial distribution by the normal distribution considering proportions figure scatter plot of control rna-seq rpkm versus infected rna-seq rpkm of significant transcripts (p < . ) with absolute fold change value of or more. most genes from both samples are closely clustered except for genes label to . and are mx transcripts and , respectively, and = rsad transcripts and , respectively, = plin and = serpinb . rather than raw counts so that it can be used reliably on libraries of different size. the transcriptional profiles of selected genes in fipv in vitro infected cells, as well as cats diagnosed with fip, were explored. the expressions of a h, which involved in viral rna and dna editing causing mutation during rna virus infection [ ] and pd- and its ligand (pd-l ), which are involved in programmed cell death and negative regulation of t cells immune response [ ] , were characterised. a h has antiretroviral activity by generating lethal hypermutations in viral genomes and is associated with increased resistance to hiv- infection in certain populations [ ] . in the case of felines, a h (but not a c) has been found to reduce the infectivity of feline leukemia virus [ ] . it is interesting to detect that the expression of a h is readily expressed at higher levels in pbmcs than in fipv infected cells (tables and ), indicating the possible involvement of the gene in antiviral activity. furthermore, the up-regulation of the gene is less, compared to pd- and pd-l in fipv diagnosed cats. in addition, the expressions of a h are significantly up-regulated in fipv infected crfk cells at h.p.i. the actual reason for this high expression of a h is unclear, but suggests that this gene is essential in restricting viral replication or forming part of the type interferon-induced innate response, since recent study has indicated the involvement of a h in restricting virus replication [ , ] . our results show that up-regulation of pd- and pd-l gene's expression in pbmcs occurred in cats diagnosed with the fip disease. in general, their expressions are correlated to each other. similar patterns were also observed in chronic fiv infection [ ] and in hiv infection in humans [ ] ; where they were associated with increasing immune dysfunction and t cell depletion. previous studies have shown that although fipv cannot infect cd + and cd + t-cells [ ] , cats infected with the virus showed t cell depletion by apoptosis resulting in an acute immunodeficiency [ ] . hence, we hypothesized that pd-l could be a mediator that mediates apoptosis of cd + and cd + t-cells, since its expression is found in a wide range of nonhematopoietic cells [ ] . furthermore, the blockade of pd-l expression was found to enhance t cell immunity and cytokine production [ ] . nevertheless, more studies are required to confirm our hypothesis. it has been established that cats infected with fipv undergo extensive tissue destruction due to inflammatory reaction [ ] . even though the study was performed on infected crfk cells, a kidney epithelial cells, it is interesting to note that transcriptome analysis of cells proposed that the inflammation process was associated with proinflammatory and th -like cytokines production, due to the up-regulation of several chemokine genes, such as ccl , cxcl , and ccl ; and genes associated with innate immune responses, such as phf , atf , and irf . further study on infection of fipv in particularly type i fipv on macrophage-like cells namely fcwf- cells and samples from fip diagnosed cats will add more value to our findings. furthermore, the down-regulation of crip (a t helper regulatory gene) ( table ) also suggests that fipv infection is associated with th response, based on a study on crip gene in mice, transgenic mice, and in murine cell line [ ] . in that study, they found that the down-regulation of crip was associated with the expression of il- , ifn-γ, and tnfα. previous studies have shown that in vitro and in vivo fipv infections were associated with tnf-α and ifn-γ expressions [ , ] . based on previous studies on the growth of fipv - in crfk cells, production of progeny virus start between and hours post inoculation and increased rapidly until hours post inoculation [ ] . in our study, we found that at hours, a few complete virus genomes that completely aligned to fipv - reference genome sequence (data not shown) has already been assembled. mx expression was up-regulated in fipv infection (table ) , similar to other rna virus infections, its role in fip pathogenesis is still unclear and requires further investigation. previous studies have shown that the expression of mx gene inhibits viral replication during various rna virus infections [ , ] . early in vitro fipv infection is also associated with a marked increase in the expression of rsad (radical sadenosyl methionine domain-containing protein ) also known as viperin (table ) . previous studies have shown viperin involvement in inhibiting viral rna and/or protein synthesis during different virus infections, such as the west nile virus, dengue virus, and hepatitis virus [ , ] . hence, further study is required to define the role of viperin in fipv replication and infection. serine proteinase inhibitor clade b member (serpinb ) is the only gene that was markedly down-regulated (table ) . serpinb functions as an inhibitor of the neutrophil serine proteases, found at inflammatory sites where the inhibition of the gene prevents tissue destruction by phagocytic cells during the virus clearance process by infiltrating neutrophils and monocytes [ , ] . thus, serpinb down-regulation could possibly be part of innate immune response in recruiting phagocytic cells for the proteolytic destruction of infected cells. besides genes that modulate t cell functions, several genes with proapoptotic and/or anti-apoptotic are also differentially regulated; thus highlighting their functions in regulating the apoptosis of virus infected cells (additional file : table s and additional file : table s ). however, the pro-apoptotic gene yap- or yap- , which was found up-regulated in fiv infection in crfk cells [ ] , was not up-regulated in this study. in conclusion, this present study has described the transcriptional profiles of cellular genes in vitro system can be applied to an in vivo situation and the possible involvement of a h, pd- , and pd-l genes during fipv infection. however, further studies are required to elucidate the roles of these genes, and their interactions with other genes, during fipv replication and infection especially in in vivo model. monolayers of crandell rees feline kidney (crfk) cells (atcc® no. ccl- ™) were grown in a base media consisting of minimum essential medium (mem), % fetal bovine serum (fbs), % penicillin and streptomycin, % amphotericin b, and non-essential amino acids at °c and % co . for transcriptome study, crfk cells were infected with fipv - (atcc® no. vr- ™) at a multiplicity of infection (moi) of . the virus was incubated for one hour for virus attachment. after incubation, % fbs mem was added and the cells were incubated for a further hours. at the end of incubation, the cells were harvested using tryple™ and centrifuged twice in a pbs at °c for minutes at rpm. cell pellets were stored at − °c until rna extraction. for the control sample, the same process was applied, with the exception that ml of sterile pbs was used to replace the virus. an rneasy® mini kit (qiagen®, usa) was used to extract and purify rna samples as per the methods recommended by the manufacturer. the quality of the extracted rna was determined by an ultrospec pro uv/visible spectrophotometer (ge healthcare, uk), where samples with an absorbance ratio value (a / a ) of . to . were considered for further analysis with an agilent® bioanalyzer. samples with rna integrity numbers (rin) to , and concentrations higher than ng/μl per sample, were sent for illumina gaii sequence analysis. a total of . gb of sequencing data, comprised of both control and infected samples, was imported into the clc bio genomic workbench (gwb). the sequences were trimmed for adapter sequences and low quality base. the trimmed raw sequences were subjected to rna-sequence analysis, by mapping them to an annotated feline genome reference sequence [ ] accounting for a maximum of two gaps or mismatches in each sequence. unpaired group comparisons, based on reads per kilo base per million (rpkm) [ ] , were chosen as expression values for comparison. kal's z test statistical analyses, based on false discovery rate (fdr) < . and fold change > . were used to filter the expressed transcripts. the resulting list was then blast at ncbi servers (http://www.ncbi.nlm.nih.gov/) using gwb's builtin blast (blastn, refseq_rna or nr databases, mammals only). homologous sequences with the lowest e-value, highest score, and lowest percentage of gaps to the query sequence, was selected as the transcript identity. in order to validate the transcriptome results, the expression profiles of genes (a h, pd- , and pd-l ) were analysed using real-time pcr. briefly, viral rna from fipv strain - infected crfk cells at , , , , , and hours post infection (h.p.i.) were collected and processed as described previously. control cells, inoculated with pbs only, were used as a control. primers were designed using primer-blast (http://www.ncbi.nlm. nih.gov/tools/primer-blast/) and synthesized by aitbiotech pte ltd (singapore) ( table ). the reactions were performed using sensifast™ sybr no-rox one step kit (bioline ltd, uk) on bio-rad cfx ™ real-time system, with c ™ thermal cycler (bio-rad laboratories, usa). briefly, the reaction mixture of μl contained μl × sensifast sybr no-rox one-step mix, . μl forward & reverse primers ( nm for gadph, pd-l and a h, nm for pd- and nm for ywhaz), . μl rt, . μl ribosafe rnase inhibitor, . μl h o, and μl extracted rna. the rt-qpcr reaction conditions were as follows; one cycle at °c for mins, one cycle at °c for mins, and cycles at °c for secs; then °c (ywhaz), °c (pd-l ), °c (gapdh), °c (a h), and °c (pd- ) for secs; and finally, at °c for secs. one cycle for the dissociation curve for all reactions was added and melting curve analysis was performed. data generated from the technical triplicate experiment was analysed with -ΔΔct method [ ] using bio-rad cfx manager version . . gapdh and/or ywhaz genes were chosen as reference genes, based on previous studies [ , ] . besides fipv infected cell cultures, peripheral blood mononuclear cells (pbmcs) were also used to analyse the transcriptome results. six fip diagnosed domestic short hair breed cats, with ages ranging from months to adult, that were admitted to university veterinary hospital, upm, were considered for this study ( table ). the cats tested negative for felv and fiv antibodies, but positive for fcov antibodies, and showed abdominal/pleural effusion. meanwhile, healthy cats, with negative results for fcov, felv, and fiv antibodies, were selected as controls. the kits for fcov, felv and fiv antibody tests originated from biogal's feline coronavirus (fcov) [fip] immunocomb® antibody test kit (biogal galed laboratories, usa) and idexx's snap® combo felv ag/fiv antibody test kit (idexx laboratories, usa), respectively. the tests were performed as per the methods recommended in their respective manuals. the health assessment and blood collection of the cats were performed by a trained and certified veterinarian (gts). the sampling were performed according to internationally recognized guidelines and recommended by the animal care and use committee at the faculty of veterinary medicine, universiti putra malaysia. two to ml of cat blood was drawn and stored at °c in bd vacutainer® (bd usa) edta-k tubes. parts of the blood were used for the test kits, while the rest was processed for pbmcs extraction. pbmcs were isolated using the ficoll-paque™ plus (ge healthcare, usa) method, according to the manufacturers protocol. total rna from pbmcs was isolated using an rneasy mini plus kit (qiagen, germany), as described by the manufacturer. rna quantity and purity was measured and assessed using a nanodrop nanophotometer p-class (implen gmbh, germany). the isolated rna samples were either kept at − °c for further analysis, or immediately used for real-time rt-qpcr analysis. additional file : table s . list of transcripts from up regulated genes with proportions fold change of or more (kal's z test, fdr < . ) with their blast results, ncbi accession number and gene product function. table s . list of transcripts from down regulated genes with proportions fold change of − or more (kal's z test, fdr < . ) with their blast results, ncbi accession number and gene product function. prevalence of feline coronavirus in two cat populations in malaysia a review of feline infectious peritonitis virus infection: - feline infectious peritonitis the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses mutation in spike protein cleavage site and pathogenesis of feline coronavirus natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease apoptosis and t-cell depletion during feline infectious peritonitis a "possible" involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis kinetic analysis of a complete poxvirus transcriptome an immediate early class of gene hepatic transcriptome analysis of hepatitis c virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance gene-expression changes induced by feline immunodeficiency virus infection differ in epithelial cells and lymphocytes viral transcriptome analysis of feline immunodeficiency virus infected cells using second generation sequencing technology a greedy algorithm for aligning dna sequences attachment and internalization of feline infectious peritonitis virus in feline blood monocytes and crandell feline kidney cells dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources functions, structure, and read-through alternative splicing of feline apobec genes increased pd-l expression and pd-l /cd ratio on dendritic cells were associated with impaired dendritic cells function in hcv infection polymorphism in human apobec h affects a phenotype dominant for subcellular localization and antiviral activity reduced apobec h variant antiviral activities are associated with altered rna binding activities feline programmed death and its ligand: characterization and changes with feline immunodeficiency virus infection upregulation of pd- expression on hiv-specific cd + t cells leads to reversible immune dysfunction pd- and its ligands in tolerance and immunity blockade of programmed death- ligands on dendritic cells enhances t cell activation and cytokine production regulation of cysteine-rich intestinal protein, a zinc finger protein, by mediators of the immune response cellular composition and interferon-γ expression of the local inflammatory response in feline infectious peritonitis (fip) replication of feline coronaviruses in peripheral blood monocytes andes virus stimulates interferon-inducible mxa protein expression in endothelial cells west nile virusinduced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral mxa protein identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections analysis of isg expression in chronic hepatitis c identifies viperin as a potential antiviral effector the neutrophil serine protease inhibitor serpinb protects against inflammatory lung injury and morbidity in influenza virus infection serpinb protects the mature neutrophil reserve in the bone marrow akt phosphorylates the yes-associated protein, yap, to induce interaction with - - and attenuation of p -mediated apoptosis the ucsc felcat data from the dec. catchrv e draft assembly mapping and quantifying mammalian transcriptomes by rna-seq analysis of relative gene expression data using real-time quantitative pcr and the (−delta delta c(t)) method a validation of feline reference genes for gene expression measurements in snap-frozen tissues quantitative taqman® real-time pcr assays for gene expression normalisation in feline tissues submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to thanks laboratory personnel at virology lab, faculty of veterinary medicine, upm and laboratory of vaccines and immunotherapeutics, institute of bioscience, upm. this project is funded by fundamental research project no: - - - fr, ministry of higher education malaysia. the funding source has no role in this study. the authors declare that we have no competing interests. msrh & cok -cell culture works; virus inoculation; rna extraction, purification and quantification; rt-qpcr assays; bioinformatic analysis; data analysis and interpretation; wrote the manuscript. gts -diagnose cats; collect blood samples from cats with fip symptoms; perform fcov, fiv and felv kit tests. tswhelps with pcr; primers design; rt-qpcr assays and data analysis. ssa, mhb & arosecure & manage fundings; coordinated the project; designed experiments and collaborated in analyzing data and writing the manuscript. all the authors have read and approved the final manuscript. key: cord- -v piprx authors: zhao, guangyu; lin, yongping; du, lanying; guan, jie; sun, shihui; sui, hongyan; kou, zhihua; chan, chris cs; guo, yan; jiang, shibo; zheng, bo-jian; zhou, yusen title: an m e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent h n influenza viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: v piprx background: a growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (hpai) h n viruses. consequently, there is an urgent need to develop an effective and safe vaccine against the divergent h n influenza viruses. in the present study, we designed a tetra-branched multiple antigenic peptide (map)-based vaccine, designated m e-map, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein (m e) of a hpai h n virus, and investigated its immune responses and cross-protection against different clades of h n viruses. results: our results showed that m e-map vaccine induced strong m e-specific igg antibody responses following -dose immunization of mice with m e-map in the presence of freunds' or aluminium (alum) adjuvant. m e-map vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. the m e-map-based vaccine protected immunized mice against both clade : vn/ and clade . . : sz/ h h n virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of h n viruses. conclusions: these results suggest that m e-map presenting m e of h n virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of hpai h n viruses. the re-emergence of h n highly pathogenic avian influenza (hpai) in has caused fatal cases among a total of infected individuals [ ] . therefore, there is an urgent need to develop safe and effective antiviral strategies for the prevention of any future pandemic of h n hpai [ , ] , among which vaccination is still the most effective means to prevent influenza a virus infection. due to current vaccine technologies facing annual problems with vaccine-strain matching, some conserved antigens of influenza a virus become promising target for the development of influenza vaccines with broad cross-protection. in comparison with other surface proteins of h n viruses, matrix protein (m ) is the most conserved. the native m protein exists as a homotetramer formed by two disulfide linked dimers, with each monomer consisting of amino acids [ , ] . the -amino-acid extracellular domain of m protein (m e) is remarkably conserved across influenza a subtypes [ ] . passively transferred anti-m monoclonal antibodies (mabs) accelerated lung viral clearance [ ] , and mabs recognizing the n-terminus highly conserved epitope in m e protected mice from lethal influenza a virus challenge [ ] , implying that m , in particular m e, may serve as an attractive vaccine target. currently, the rapid evolution of h n virus and the co-circulation of multiple antigenic variants in multiple regions determine that development of h n vaccine with cross-protection against divergent h n viruses would be inevitable. although a number of m e-based vaccines have been reported to provide broad-spectrum protection against ordinary human influenza virus infection [ ] [ ] [ ] [ ] [ ] , the cross-protective effect to divergent h n viruses was undocumented. in the present study, we designed and synthesized a tetra-branched multiple antigenic peptide (map) derived from the m e sequence of h n virus vn/ strain, denoted as m e-map, with an aim to develop a m e-based vaccine for induction of m e-specific immune responses and cross-protection of the vaccinated animals against lethal challenge of divergent h n virus strains. to evaluate humoral immune responses potentially induced by m e-map, mice were vaccinated with μg of m e-map plus freund's or aluminium (alum) adjuvant as described in methods, and m e-specific igg antibodies were detected in mouse serum samples by elisa. as shown in figure , m e-map induced strong m e-specific igg antibody responses, with the highest titer reaching : and : for m e-map+fca/fia and m e-map+alum, respectively, at days post last boost vaccination. in contrast, only background level of antibody responses was detected in the mice receiving adjuvant alone. two phylogenetically distinguished h n virus isolates, clade : vn/ and clade . . : sz/ h were selected to evaluate the protective immunity afforded by m e-map vaccine in vivo. two weeks after the last boost, mice were challenged with ld of h n virus vn/ or sz/ h. five days post-challenge, lungs were removed from infected mice, and infectious virus titers in the lung tissues were measured to determine the protective effects of m e-map vaccine on viral clearance. compared with those from the adjuvant control (fca/ fia or alum), the virus titers in the lungs of m e-map vaccinated mice were significantly lower after h n virus challenge (p < . ) (figure ), suggesting that m e-map vaccine can induce protective immunity against viral replication in vaccinated mice. further examination of the lung tissues of virus-challenged mice revealed dramatic histopathological damage in the pulmonary airways and parenchymal tissues in the adjuvant group, including severe damage of bronchial epithelium with necrosis and desquamation, pulmonary vascular dilatation and congestion, infiltration of prominent number of lymphocytes accompanied by exudates and severe edema, especially around vessels, as well as broadening interstitial spaces or fused alveoli walls with focal hemorrhage ( figure a ). in contrast, lungs of m e-map-vaccinated mice exhibited less figure m e-specific antibody responses induced by m e-map vaccine. mice were vaccinated with m e-map plus fca/fia (s.c.) or alum (i. m.) adjuvant for a total of times. mice receiving fca/fia or alum alone were served as adjuvant controls. mouse sera were collected preimmunization and days post-each immunization for detection of m e-specific antibodies by elisa. the end-point titer of each sample was determined as the highest dilution that yielded an od nm value greater than twice that of similarly diluted serum sample collected prevaccination. the data are expressed as geometric mean titer (gmt) ± standard deviation (sd) of mice per group. the lower limit detection ( : ) is indicated by a dotted line. experiments were repeated three times. histopathological changes, accompanied by only mild pulmonary interstitial pneumonia and moderate lymphocytic infiltration ( figure b ). the above data implied that m e-map vaccination may protect the mice against lethal challenge of divergent h n viruses through a combination of limiting viral replication in the lungs and attenuating virus-induced lung pathology. after receiving the lethal dose ( ld ) of two h n virus strains, the m e-map vaccinated mice were further evaluated in terms of cross-protective ability by daily observation of the clinical symptoms, including weight loss and survival rate for two weeks, and then histopathological examination following removal of lung tissues. from day after vn/ and day after sz/ h virus infection, the adjuvant control mice (fca/fia or alum) developed obvious clinical signs, including ruffled fur, hunched posture, rapid breathing, inactivity and paralysis of posterior limb. these clinical signs were either not observed or were delayed for - days in the m e-map vaccination group. most m e-map vaccinated mice with clinical signs only exhibited slight or partial piloerection for - days and then recovered. moreover, compared to the adjuvant control, m e-map vaccinated mice that did not survive the challenges were observed to have delayed the onset of illness and longer survival days. the observed protection against clinical signs correlated with the changes in body weight. in the adjuvant group, body weight dramatically decreased and even reached a near % severe weight loss after h n virus infection. in contrast, the average body weight in the m e-map vaccination group only slightly decreased (less than %) during - days after challenge and then steadily increased ( figure a ). all control mice receiving fca/fia or alum adjuvant alone died from virus challenge, while % to % of the mice vaccinated with m e-map survived the lethal h n virus challenge ( figure b ), with significantly increased survival rate (p < . ). at days postchallenge, lung tissues from surviving mice were removed to evaluate viral replication and histopathological damage. no h n virus was detected in the lungs of surviving mice, and the lung tissues of these mice presented almost normal structures (data not shown). the above data confirmed that m e-map vaccine can afford cross-protection against lethal challenge of divergent h n viruses. a cross-protective vaccine for antigenic variants of h n virus is a very important component in prophylactic strategy against a possible human pandemic. , and lungs were collected for detection of virus titer five days later. the data are expressed as log tcid /g of lung tissues. the lower limit of detection is . log tcid /g of tissues as indicated by a dotted line. the data are presented as gmt ± sd of mice per group. * indicates p < . compared to the fca/fia control group; ** means p < . compared to the alum control group. experiments were repeated three times. although the highly conservative m e of influenza a virus is one of the most promising target for development of universal influenza vaccines, some strategies would be required to improve immunogenicity of vaccines based on m e containing only amino acid. the high molar ratio and dense packing of multiple copies of a target antigen in map system have been shown to stimulate better immune responses than single-chain peptides [ ] [ ] [ ] . mozdzanowska et al. [ ] reported that map system presenting human influenza m e sequence was an effective immunogen, with anti-m e igg antibodies from mice immunized with m e-map binding specifically to m -expressing cells. however, it is unclear whether m e-map could be used as a vaccine candidate to provide effective protection against hpai h n virus. different from human influenza a viruses of h , h or h , h n viruses have circulated only in domestic and wild birds so far [ ] [ ] [ ] [ ] . the h n viruses leading to human infections still belong to the avian type [ ] . m e vaccine designed on sequences of human influenza virus h , h or h subtype may not provide the same protection against h n virus leading to human infections [ ] . moreover, the - amino acid region of m e was consistent with host restriction specificities [ ] . in this study, we designed m e-map in tetrabranched form containing copies of sequence overlapping m e of vn/ , a hpai h n strain ( figure ). high titers of m e-specific antibody responses could be induced following immunization of m e-map plus freund's adjuvant, a commonly used adjuvant in animal experiments or alum, a common adjuvant for human vaccines (figure ). although it has been shown that m e-specific igg antibodies do not exhibit an ability to directly neutralize virus in vitro, the antiviral effect of m e-based vaccines was mediated by antibodies to m e antigen. the mechanism of such antibody mediated antiviral effect could be due to antibody-dependent cellmediated cytotoxicity (adcc) and/or complementmediated cytotoxicity (cdc) [ , ] . therefore, the induction of m e-specific antibody responses was necessary for m e-based vaccines in the prevention of h n virus infection. strikingly, m e-map vaccination with freund's and alum adjuvants conferred cross-protection against a lethal challenge with not only the homologous strain, vn/ in clade , but also a divergent strain, sz/ h in clade . . . m e-map vaccination in mice delayed the onset of illness, prolonged survival, alleviated body weight loss, limited viral replication and restricted histopathological damage in lung tissues (figure , figure and figure ). the clade categorization of the vn/ and sz/ h strains was based on the phylogenetic analysis of the ha gene. the clade . . viruses seem to have been prevalent in china since late and were also responsible for human infection in laos, malaysia and vietnam [ , ] , which included a/hunan/ / , one of the most recent human isolates from china [ ] . compared with the monomeric m e peptide-based vaccines, m e-map vaccine could induce stronger protective immunity due to the immune enhancing effect of the t-helper epitope in the vaccine [ ] . in terms of the safety, the synthetic peptide-based m e-map vaccine is superior to the dna-, adenoviral vector-, and recombinant protein-based m vaccines. for example, dna vaccines have the potential to integrate the viral genomes into human dna, to develop autoimmunity, and to induce antibiotic resistance [ ] . the recombinant protein-based vaccine may have contamination of endotoxin and other unwanted antigens from cell cultures [ ] . in spite of a relatively higher cost of production, synthetic peptide-based vaccine can be rapidly designed and synthesized and more convenient for storage and transportation, making m e-map vaccine a better choice for speed development of effective and safe vaccines to combat emerging influenza pandemic. based on the evidence of our findings, the approach of developing m e-based vaccines with broad cross-protection is feasible. some strategies, such as incorporation of appropriate adjuvants into the present map system, modification of the present m e consensus sequences of h n viruses as vaccine target, and/or selection of other m e expression forms, might be helpful to achieve full cross-protection against lethal h n virus infection. the effects of such approaches need to be determined in further studies. the data reported here support the concept of developing an m e-based vaccine candidate that could provide cross-protection against divergent hpai h n viruses. such vaccine based on the highly conserved target antigen of h n viruses might be used in combination with current h n influenza vaccines to enhance protection and to prevent a possible future human pandemic of h n influenza. in the present study, we synthesized tetra-branched m e-map based on the m e sequence of h n virus. the m e-map vaccine induced strong m e-specific igg antibody responses, being able to protect mice against lethal challenge of both clade : vn/ and clade . . : sz/ h h n viruses. based on the evidence of our findings, the approach of developing m ebased vaccine with broad cross-protection is feasible. the data reported here support the concept of figure shows the composition and sequence of m e-map. female balb/c mice at - weeks were purchased from the laboratory animal unit and housed in the animal facility of the university of hong kong following the approved animal care protocols. mice were rested for weeks before immunization. the animal study was approved by the department of health of government of hong kong special administration region, and university animal ethics committee of the university of hong kong. hpai h n virus isolates used in this study were clade : vn/ and clade . . : sz/ h. h n viruses were grown in the allantoic cavities of -day-old embryonated chicken eggs. virus-containing allantoic fluid was harvested and stored in aliquots at - °c until use. the ld of each virus stain was determined in mice after serial dilutions of the virus stock. all infectious experiments related to h n viruses were performed in an approved biosafety level (bsl- ) facility at the university of hong kong. mice were subcutaneously (s.c.) prime vaccinated with m e-map ( μg/mouse) plus freund's complete adjuvant (fca, sigma) and boosted twice with the same amount of immunogen plus freund's incomplete adjuvant (fia, sigma) at -week intervals. for parallel experiments, mice were also intramuscularly (i.m.) vaccinated with the same amount of m e-map immunogens plus alum adjuvant (sigma) at the same condition of the above. mice injected with freund's or alum adjuvant were used as the respective control. mouse sera were collected before immunization and days post-each vaccination for detection of antibody responses. two weeks post-last vaccination, mice were intraperitoneally (i.p.) anesthetized with ketamine-xylazine ( / mg/kg), and intranasally (i.n.) challenged with ld of clade : vn/ or clade . . : sz/ h h n virus stain. infected mice were observed and weighed daily for weeks. lung tissues were collected from euthanized mice days post-challenge for further virological test and histopathological analysis. the m -specific igg antibody in the vaccinated mouse sera was detected by elisa as previously described [ ] with some modifications. briefly, -well microtiter plates were pre-coated with μg/ml of m e-map overnight at °c. after blocking with % bsa containing . % tween- in pbs, serial diluted mouse sera were added to the plates, followed by adding hrp-conjugated rabbit anti-mouse igg ( : , , invitrogen, carlsbad, ca) for h at °c. assay was developed using , ', , 'tetramethylbenzidine (tmb) (zymed, carlsbad, ca), and the reaction was stopped by adding n h so . the absorbance at nm was measured by an elisa plate reader (sunrise™ microplate reader, tecan, nc). lung tissues from euthanized mice were aseptically removed and homogenized in minimal essential medium (mem) plus antibiotics to achieve % (w/v) suspensions of lungs. ten-fold serial dilutions of samples were added in quadruplicate to the monolayer of madin-darby canine kidney (mdck) cells seeded at -well cell culture plates h before infection, and allowed to absorb for h at °c. fresh medium was then added to the cells and continued to incubate for h. virus cytopathic effect (cpe) was observed daily and the viral titer was determined by the hemagglutinin (ha) test indicated as follows. fifty microlitre of . % turkey red blood cells (lampire biological laboratories, pipersville, pa) was added to μl of cell culture supernatant and incubated at room temperature for min. wells containing ha were scored as positive. the virus titer was calculated by the reed and muench method and expressed as log tcid /g of lung tissues. the lung tissues of challenged mice were immediately fixed in % neutral buffered formalin and embedded in paraffin wax. sections were made at - μm thickness and mounted on slides. histopathological changes were examined by h&e staining and observed under light microscopy as previously described [ ] . the significance between survival curves was analyzed by kaplan-meier survival analysis with log-rank test. other data were analyzed using the -tailed student's t test. p < . was considered significant. all analyses were performed in graphpad prism software. cumulative number of confirmed human 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adenoassociated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection an m e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent h n influenza viruses authors' contributions gz, bz and yz designed research. gz, yl, jg, ss, hs and cc performed research. gz, ss, zk and yg analyzed data. gz, ld, sj, bz and yz wrote and modified the paper. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - fvbshz authors: balakrishnan, krishnan nair; abdullah, ashwaq ahmed; bala, jamilu abubakar; jesse, faez firdaus abdullah; abdullah, che azurahanim che; noordin, mustapha mohamed; mohd-azmi, mohd lila title: multiple gene targeting sirnas for down regulation of immediate early- (ie ) and dna polymerase genes mediated inhibition of novel rat cytomegalovirus (strain all- ) date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: fvbshz background: cytomegalovirus (cmv) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. in the present study, with the aim of exploring new strategies to counteract cmv replication, several anti-cmv sirnas targeting ie and dna polymerase gene regions were characterized and used as in combinations for antiviral therapy. methods: the rat embryo fibroblast (ref) cells were transfected with multi sirna before infecting with cmv strain all- . viral growth inhibition was measured by tissue culture infectious dose (tcid ), cytopathic effect (cpe) and droplet digital pcr (ddpcr) while ie and dna polymerase gene knockdown was determined by real-time pcr. ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual sirnas. results: there was no significant cytotoxicity encountered for all the combinations of sirnas on ref cells analyzed by mtt colorimetric assay (p > . ). cytopathic effects (cpe) in cells infected by rcmv all- had developed significantly less and at much slower rate compared to control group. the expression of targeted genes was downregulated successfully resulted in significant reduction (p < . ) of viral mrna and dna copies (dpb + dpc: %, %; dpb + ie b: %, %; dpb + dpc + ie b: %, %). flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined sirnas-treated cells compared to control group. notably, the sirnas targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. conclusions: in conclusion. the study demonstrated a tremendous promise of innovative approach with the deployment of combined sirnas targeting at several genes simultaneously with the aim to control cmv replication in host cells. cmv often considerate as silent public health burden usually does not produce any symptoms after the infection. the prime reason was due to the ability of the virus to enter asymptomatic infection, once the virus replication was controlled by the host immune system [ , ] . however, cmv can cause life-threatening condition in immunocompromised patients whom undergo transplant procedures, individuals diagnosed with aids and injured with third degree burn and elderly people to some extent [ ] [ ] [ ] . sadly, immature immune system of unborn child is prone to cmv infection causing complications such as microcephaly, mental retardation and hearing loss [ , ] . the term rnai is generally understood as a mean of evolutionary conserved mechanism during post-transcriptional silencing triggered by double stranded rnas, in wide range of eukaryotic organisms. this system was initially discovered in nematode caenorhabditis elegans [ ] . slowly, the usage of rnai turned to be a broader wave of advance which has been employed as a treatment method for many infectious diseases, cancerous and nerve dysfunction disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] . introducing synthetic - nucleotides of sirna could specifically inhibit the cellular mrna in order to control the gene expression [ ] . up to date, various studies have been reported that delivering sirna, to inhibit the viral replication and gene expression of growing number of human infectious viruses were promising [ , ] . it was suggested that a greater focus on sirna could produce interesting findings that account more for the sirna utilization as antiviral strategy for future therapeutics. growing research work proves that rnai can inhibit or suppress most of the viruses despite rna or dna and whether single or double stranded structure. combination of rnai targeting different conserved regions is expected to function efficiently in reducing the chances of viral escape. for instance, in other mono-therapies, sequence specific rnai facing problem where the virus escapes due to the mutant in target region resulting in growth of viral mutant [ ] [ ] [ ] . this statement validated by few studies such as polio, hiv and hcv where the viral activity of single sirna reported to decrease in efficiency [ ] [ ] [ ] . viruses evolve to escape from host rnai by encoding "suppressors of rnai silencing" (srs) which is another disadvantage of single use of sirna. sirna cocktail targeting different region of viral genome lowers the chances of viral mutant formation and prevent viral escape [ ] [ ] [ ] [ ] . nevertheless, besides virus infections, sirnas can be utilized to silence multiple genes at a time which could benefit in treating brain tumorigenesis involving multiple genetic alterations and also targeting a suitable apoptotic gene as a treatment [ , ] . in addition, the availability to maintain the wide array of cells including islets of langerhans could provide a strong foundation to use sirnas for diabetic studies [ , ] . therefore, exploring combinations of sirnas as a potential therapeutic approach for malaysia cmv isolate has gear up for the treatment destination [ ] . interestingly, this local strain (rcmv all- ) has been identified crossing the placenta infecting the pups making it suitable for congenital infection on animal model [ ] . the availability of the complete genome sequence of this virus add on more advantages for pathogenicity study and antiviral development [ , ] . therefore, this study was conducted to screen combinations of sirna for their effectiveness on inhibiting rcmv all- virus replication and gene expression. the sirna combinations were selected based on the successive percentage of individual sirna inhibiting rcmv all- replication and gene expression (data not shown). the tested sirna combinations were dpb + dpc, dpb + ie b, dpc + ie b and dpb + dpc + ie b where dpb and dpc sirnas targeting dna polymerase region while ie b targeting immediate early region. the sequences of designed sirnas were displayed in table . rat embryo fibroblast cells were propagated in % fetal bovine serum supplemented with dmem nutrient solution. the cells were maintained with at % co and °c in an incubator. prior to sirna transfection, the cells were seeded on well plates to achieve - % of confluency level. after h, combination sirnas were transfected together with lipofectamine transfection reagent ( . μl/well). in each well, sirnas were mixed to produce a final concentration of pmol/well. appropriate controls for treatment and non-treatment were included as well followed by twenty-four hours of incubation. meanwhile, during the incubation, dmem media without antibiotic was replaced at h post transfection to avoid any contamination. next, cells were infected with rcmv all- isolated from uterus and placenta of rats [ ] at moi of . the plates were incubated for h and then the cells were washed with cold pbs followed by addition of fresh dmem medium supplemented with % serum. the cells were then monitored continuously for cpe occurrence and the cultured cells were collected for all the analysis at appropriate time points. ganciclovir was used as positive control and scramble sirna as negative control for treatment groups while untreated cells as positive control and uninfected cells as negative control for non-treatment groups. each sirna combination was duplicated and was performed twice independently. cells with optimized density, × were seeded in wells plate supplemented with dmem media with % fbs. the cells were incubated at °c for h. once the cells are established for the treatment, the media was discarded, and the confluent cells were washed with cold pbs. the combination sirnas was transfected and incubated at °c without the infection of the virus. the plates were removed from incubator at , and h of time interval. then, mtt assay was carried out to identify the cell viability. all the cells were washed twice with pbs followed by addition of ( mg/ml) mtt (tetrazolium dye, -[ , -dimethylthiazol- -yl]- , -diphenyltetrazolium bromide) reagent and incubated for h at ºc. the mtt addition procedure was taken place in dark area and the plate was covered with aluminium foil during the h incubation due to the sensitivity of mtt towards light. later on, the mtt reagent was discarded carefully, and μl of dimethyl sulfoxide was added to each well. then, the plate was covered again with aluminium foil and leave for min at room temperature. this is to make sure the formazon dye crystals to dissolve completely into purple color. lastly, the results were obtained from the measured absorbance at nm using a spectrophotometer microplate reader. each assay was performed in triplicates and the cytotoxicity was calculated from an average of replicates. the final results are presented as mean ± standard deviation. tcid was conducted to investigate the effects of combination sirnas on controlling rcmv all- titer at different time intervals of post infection (day and ). following the transfection of combination sirnas, the ref monolayers were infected with moi rcmv all- . tcid of each combination sirnas transfected samples were analyzed and compared with tcid of non-transfected virus control group as previously reported [ ] . all treatments were performed in triplicate and the results were expressed as mean ± se. flow cytometry technique was employed to evaluate the percentage of viable cells, early apoptosis cells; late apoptosis cells and necrotic cells of combinations sirna treated and control groups. the samples were prepared by annexin v-fitc apoptosis detection kit (nacalai tesque, japan). first, the cells were washed twice using cold pbs followed by trypsinization to detach the cells from the culture plates. briefly, × cells/ml were resuspended in annexin v binding solution ( ×). then, the µl of suspension cells were incubated with µl of annexin v-fitc conjugate and µl of propidium iodide (pi) at room temperature. after min, µl of annexin v binding solution ( ×) was added to the sample solution and were analyzed with a facscalibur flow cytometer (bd, bioscience, usa). droplet digital pcr is an emerging third generation of nucleic acid detection methodology gaining an interest for absolute quantification of any target sequence without the needs of standard curve. before the commencement of ddpcr experiment, primers and probe were designed targeting dna poly region and were synthesized by idt technologies following standard procedures. selected primer sequences were ccg aag tac cag att caa (forward primer), gac gag agg gag tat att (reverse primer) and fam-cgg acg gtg aac tcg ttt tt-tamra (taqman probe). the experiment began with the preparation of reaction mix at µl per reaction. after that, . µl of each forward and reverse primers are added into the reaction mix followed by . µl of probe. the samples were labelled as shown in table . then, . µl of dnase-free water was added. all components were then mixed up properly by pipetting up and down. equally aliquot which was µl were dispensed into each reaction tube. then, µl of dna sample are added accordingly into the pcr tubes. the reaction were again mixed properly and be allowed to be equilibrated at room temperature for about min. once the reaction mixtures were ready, µl of the pcr reaction mixture were then loaded into the sample well of a dg ™ cartridge as well as µl of droplet generation oil into the oil wells. the cartridge loaded with sample was placed inside the q × droplet generator to partition the sample hence generating the droplet for min. then, the obtained droplets were carefully transferred into a clean -well plate. the plate was then sealed with foil by using the p × pcr plate sealer. pcr thermal cycling was then proceeded, and the sealed -well plate was placed in the q × droplet reader in order to detect the positive and negative droplets. quantasoft ™ software was used to analyze the obtained results. the replication of virus can be identified indirectly by physical appearance changes of normal cells. it is also known as cytopathic effect (cpe) monitoring. in order to determine the efficacy of combination sirnas, the transfected wells were monitored for virus induced cpe out to days post infection by a using nikon eclipse ts inverted microscope (nikon instruments, inc., new york, usa). all the images of combination sirnas transfected as well other control groups were compared and recorded. gene expression study of combination sirnas treated samples were carried out using optimized custom designed taqman probes via cfx tm real time pcr detection system (bio-rad, usa). since combination sirnas having two different gene targets: ie and dna poly, each combination sirnas were analyzed for both genes' expression. all the primers and probes have been optimized and appropriate standard curve have been generated (additional file : figure s ). rcmv all- mrna was extracted from the control and sirna treated samples following manufacturer instruction by using a genezol ™ trirna pure kit (geneaid, uk) with slight modifications. the rna was finally eluted in µl of rnase-free water and was maintained on − °c for further analysis. the quantity and quality of extracted rna were assessed by spectrophotometry using the nanodrop spectrophotometer (thermo scientific ™ ). next, complementary dna was synthesized using tetro cdna synthesis kit (bioline, uk) on thermal cycler (biorad, usa) following manufacturer's instructions. the prepared cdna was stored at − °c for subsequent analysis. finally, gene expression study of combinations sirnas treated samples were carried out using designed taqman probes via cfx ™ real time pcr detection system (bio-rad, usa). specific primers and probe were used for each sirna region as depicted in table . to normalize the expression analysis, glyceraldehyde -phosphate dehydrogenase (gapdh) was used as housekeeping gene which served as internal control to quantify the mrna expression in all the combination sirnas treated and non-treated samples. the reaction was set up with cdna as template with nm concentration of each specific forward and reverse primers, nm of probe, × sensifast probe hi-rox mix and appropriate nuclease free water. the cycling conditions was followed as for standard curve generation (data not shown) mean quantitative cycle values (cq) were obtained from the triplicates and recorded using bio-rad, cfx manager software version . (bio-rad, usa). four combination sirnas: dpb + dpc, dpb + ie b, dpc + ie b and dpb + dpc + ie b were assessed for their effectiveness on inhibiting the ie and dna poly gene expression of rcmv all- . the relative fold change of the genes treated with sirna were compared with untreated control group and the relative fold changes were calculated using − ΔΔct method. bio-rad, cfx manager software version . (bio-rad, usa) was used for the normalization of target region with house-keeping gene gapdh and quantification of fold changes were calculated as described previously [ ] . the percentage of knockdown efficiency was calculated following − ΔΔct method with slight modification [ ] . the levels of sirna treated target expression was normalized to non-target gapdh as reference gene (ref) within the same sample as equation below: next, the Δct for each biologically replicate was transformed exponentially to the Δct expression followed by determining the standard deviation. the equation as follows: then, the mean was normalized to the expression of sirna treated sample with non-targeting sirna sample to find Δct expression level. percentage of knockdown was calculated as equation below: drug resistances are important issue to be addressed where virus has the capability of developing it. in this combination treatment, the ability of rcmv all- to develop drug resistance via mutation occurrence in target regions was investigated. after the sirna combination transfection, rcmv all- was infected and the extracellular progeny virus was harvested after days pi. the harvested virus was used to re-infect the ref cells after being transfected again with sirna combinations. after days pi, the virus was harvested, and this method was repeated up to infections. the final harvested virus was subjected for nucleic acid extraction together with stock of virus. upon confirmation, the pcr products were sent for sequencing and the obtained results were compared for sequence identity using clustal omega online software. all statistical analyses were performed using graph-pad prism version (graphpad software, usa). p values < . were considered significant. results are expressed as mean ± sd from a representative experiment performed in triplicate. combinations of sirnas were subjected for cell viability assay using mtt cell proliferation method. cytotoxicity analyses of tested sirna combinations demonstrated that cell viability was almost unaffected at an optimized concentration of pmol at different time points. however, the triple combinations dpb + dpc + ie b exhibit % toxicity level at and h for pmol. on the other hand, all the double sirna combinations such as dpb + dpc, dpb + ie b and dpc + ie b showed more than % of cell viability at highest concentration pmol (additional file : figure s ). rcmv all- was silenced with double or triple combinations and the virus titers were quantified by tcid . the reading of viral titer was recorded at two different days: and (fig. ). this is because no significant viral load was detected at day and maximum growth of virus could be achieved at day ( fig. ) . overall, different combinations sirna showed different level of virus inhibiting capacity which sirnas targeting dna polymerase showed better rcmv all- inhibition rate. administration of the sirnas before rcmv all- prevented the progression of the infection, since virus titers did not increase exponentially from day pi to day pi. by contrast, virus titers increased significantly from day pi to day pi in negative control sirna and untreated groups. for instance, dpb + dpc ( . log tcid /ml, . log tcid /ml) showed better viral inhibition rate at day and , respectively followed by dpb + ie b ( . log tcid /ml, log tcid /ml) and dpc + ie b ( . log tcid /ml, . log tcid /ml). all these combinations had similar viral titer compared to positive control ganciclovir . log tcid /ml at day p.i and . log tcid / ml at day p.i. therefore, the differences among the sirna treated and ganciclovir are insignificant (p > . ). however, triple combinations dpb + dpc + ie b ( . log tcid /ml, . log tcid /ml) displayed a lowest rate of viral inhibition compared to other combinations and gcv at both day and . on the other hand, negative control sirna and untreated groups showed higher viral titer than sirna treated groups: log tcid /ml at day p.i and log tcid /ml at day p.i. analysis of rcmv all- infected cells undergoing apoptosis/necrosis upon combination sirnas treatment were studied using flow cytometry. dot plot view illustration was used to interpret the results of ref cells (additional file : figure s ). as revealed in fig. , the percentages of viable cells are lesser compared to early apoptotic cells for all the sirnas and gcv treated groups. compared to untreated control group, dpb + dpc has significant number of viable cells and early apoptotic cells . % ± . and . % ± . respectively. the significant number of cells recorded in early apoptotic were all the combination sirnas and gcv treated group where dpb + ie b having the highest early apoptotic cells ( . % ± . ). in addition, all the sirnas and gcv treated groups showed least number of late apoptotic cells compared negative control sirna and untreated groups. meanwhile, lesser cells ( . - %) were observed for necrotic phase for all the treated groups including the control groups. the quantification of rcmv all- dna copies were presented per µl in the -fold diluted sample. therefore, the total viral dna copies/µl from stock template was calculated and presented in table . visualization of ddpcr analysis plot having positive and negative droplets were screened prior to viral dna quantification (additional file : figure s ). the final results have revealed that out of four combination sirnas, dpb + dpc has tremendously reduced the viral titre with , dna copies/µl with % of reduction compared to untreated control group having , dna copies/µl. as expected, both of these sirnas targeting dna polymerase gene region showed better viral inhibition rate compared to combination sirnas targeting ie gene region. next, dpb + ie b followed by dpb + dpc + ie b limits the virus particles with % and % rate effectiveness respectively (fig. ) . however, dpc + ie b showed to have the least effectivity rate % with , dna copies/µl. in comparison with other sirna treated groups, commercial drug gcv treatment was found to be median effective rate in reducing the viral dna copies ( , copies/µl) with % of efficiency. only % of difference in dna copies reduction percentage was encountered between dpb + dpc + ie b and gcv treatment groups. overall, the percentage of viral reduction was apparently moderate for double and triple combination sirnas and interestingly, triple sirnas combination displayed lesser inhibition rate than double combination (dpb + dpc and dpb + ie b). on the other hand, untreated control group is having high number of viral particles ( , copies/µl) due to the absence of any form of treatment. to study the cpe rate of rcmv all- in combination sirnas treated and non-treated groups, ref cells were transfected with pmol combination sirnas followed by viral infection, subsequently the cells were monitored for morphological changes at day . cytopathic effect is a reliable method to monitor the structural changes of host cells resulted from cmv invasion and propagation. there are distinct differences between cmv infected and non-infected groups. normal cells retain their fibroblast shape, no plaque formation and tightly stuck to the plate up to day (fig. b) . in contrast, the cells infected with rcmv all- exhibited evident morphological changes, starting from cell ballooning, cytoplasmic extension, shape changed to more elongated and finally forming plaque by detaching from flask. the continuation of plaque expands and achieves advanced cpe where most of the cells died and detach from the flask as shown in fig. a . in order to assess the effectivity of combination sirnas, all the treated cells were examined at day and the rate of cpe development was recorded. as expected, all the combination sirnas showed to protecting cells from rcmv all- , however, the protection degree was differing from one and another. out of four combinations, dpb + dpc shown to be most potent combination sirnas where least rate of cpe was recorded (fig. a) . at day , pmol of dpb + dpc was found to starting cpe with cell ballooning indication as showed in same figure. in comparison, negative control sirna and virus control cells started to exhibit significant advanced cpe formation at day . followed by dpb + dpc, dpb + ie b and dpb + dpc + ie b showed a good inhibition of cpe at day (figs. and ). contrary to expectations, the results did not show a constant finding in all the sir-nas where dpc + ie b sirnas are less effective and displayed similar rate of cpe development with negative control sirna and virus control cells proving their incapability in providing protection for host cells against rcmv all- . these findings showed that triple combination sirnas are not effective as double combination to inhibit the cpe rate. thus, this proves the misconception theory of conveying more sirnas the percentage of rcmv all- dna copies reduction compared to untreated control group combination providing better viral inhibition rate. the difference in results may be explained by the fact that sirnas targeting different region of genome having different rate of cpe inhibition effectiveness. taking into consideration as positive treatment control group, commercial drug gcv exhibited better rate of cpe inhibition (fig. ) compared to other combination sir-nas but lesser efficient than dpc + ie b sirnas. in order to understand more on the role of each combination sirnas during rcmv all- infection, all the four combination sirnas targeting different regions were analyzed individually with specific primers. overall, the results revealed that each combination sir-nas showed different degree of mrna reduction with dpb + dpc, dpb + ie b and dpc + ie b showed a greater reduction compared to virus control group (fig. a) . sirna dpb + dpc found to be very effective by reducing the mrna expression of dna polymerase up to % (p < . ), however % for ie gene region. this is mainly due to the sirnas itself where both sirnas and dna polymerase for all the combination sirnas. fascinatingly, between the two genes, sirnas targeting dna polymerase found to have relatively lower mrna expression compared to ie . however, if compared to commercial drug gcv, the combination sirnas were found to be less potent on inhibiting the ie and dna polymerase expressions as shown in fig. a . in comparison, triple sirnas combination showed less mrna inhibition ability on reducing the gene expressions compared to double sirnas combinations. the silencing effects of sirnas combination were further transformed to determine the percentage of knockdown efficiency as shown in table and fig. b . since the combinations are made up from different sirnas targeting different regions, each combination were assessed for both ie and dna polymerase genes knock down separately. for ie gene region, dpb + ie b and dpc + ie b combinations showed better knocking down efficiencies - % compared to dpb + dpc and triple combination dpb + dpc + ie b ( - %) respectively. on the other hand, for dna poly gene region, all the double sirnas combination showed better knocking down percentage compared to ie region where the range percentage range were between and %. the dpb + dpc combination targeting dna polymerase alone showed best level of knocking down among sirnas followed by dpc + ie b and dpb + ie b. this clearly shows that the designed sirnas were found to working best on inhibiting dna polymerase mrna expression compared to ie . noticeably, triple combinations having dna poly sirnas and one ie sirna (dpb + dpc + ie b) possess lesser potent on reducing the mrna expression thus having a moderate knocking down efficiency compared to double sir-nas combination. similar findings were also observed for viral quantification as well cpe rate observation. therefore, these findings have proven again that more sirnas in a combination not necessarily possess better knocking down efficiencies of a certain gene. besides that, gcv still considered as better option in knocking down the both dna polymerase and ie gene regions compared to rest of the sirnas combination (table ) . another important finding was the knockdown efficiency of negative control sirna. it was found to be zero percent of knocking down efficiency which are similar with virus control group, thus proving its nature as non-target control, which do not carry any impact on virus and host cells. in conclusion, each combinations sirna hold different degree of gene knock down efficiency with dpb + dpc found to be high potent to knock down dna polymerase gene while dpb + ie b for ie gene. it is common for viruses to develop resistance against treatment that subjected continuously over a certain period of time. to investigate the tendency to develop resistance via mutation by combination sirnas treated rcmv all- were harvested. then, the extracellular progeny virus was infected repeatedly up to times on combination sirnas treated ref cells. the final progeny virus was harvested and subjected for dna extraction. the entire extracted dna were sequenced by sanger sequencer platform to identify any possible mutation occurrence and compared with stock rcmv all- solution using clustal omega online software system. for every combination sirnas treatment, every region involved by the particular individual sirna was sequenced to obtain all the possible occurrence of mutation. remarkably, no any mutation was observed for all the sirnas treated regions thus, proving the ability of designed sirnas to not developing or delayed the development of sirnas resistance by rcmv all- . currently, passages of sirna treated rcmv all- were sequenced and more passages needed to be sequenced in future to identify any possible mutations. since rcmv all- is double stranded dna virus, it was expected that the rate of mutation occurrence is extremely lower than rna viruses which having higher rate of mutation incidence. this statement was supported by findings of this study where sirnas treated rcmv all- having % sequence homology with stock of virus (progeny virus without sirnas treated) proving the lower rate of mutation existence. the detailed information of aligned sequences for each sirna treated regions compared with stock viruses were portrayed in fig. . while no approved vaccine or effective therapeutic treatment for cmv, great efforts on research are being made to treat the virus infection by different strategies [ ] [ ] [ ] . the effort of making suitable vaccine for cmv started on early as reported by current national academy of medicine united states on the publication of a vaccine priority document [ ] . fascinatingly, cmv was given highest priority on the list and many strategies were implied to develop a suitable one by many biotechnology companies [ , ] . unfortunately, effective vaccines are still lacking and haven't approved any by fda. during the search of effective treatment for cmv, idea of controlling the disease by means of inhibiting virus replication and gene expression by combinations of two and more sirnas had been explored in this study. combination therapies have been tested before targeting other viral infections such as influenza, coronavirus, respiratory syncytial virus, hepatitis b&c, vaccinia virus [ , , [ ] [ ] [ ] [ ] . it was hypothesized that targeting two different genes simultaneously could silence the expression of crucial viral genes thus; act as an antiviral therapy against cmv. there are many reasons to pick sirna as a choice of potential treatment against cmv. first, they are easy to design and cheap to be produced [ ] . next, they are highly specific which can target almost any viral or cellular gene, and their stability can be increased by lyophilization [ ] . an additional advantage was they can be delivered in combinations with many sirnas or with other drugs targeting distinct regions in the same or different genes [ ] . undoubtedly, the mode of treatment action can be expanded by limiting the emergence of treatment resistance candidates [ ] . therefore, all these factors fueling up sirna research and their successive clinical trials had increased remarkably in recent years [ ] [ ] [ ] . most of the treatments works fine on early stages found to be ineffective over certain period of time. this is due to the capability of viruses to develop resistance in order to escape the attack from antiviral therapies [ ] . to resolve this, combination of sirnas targeting similar or different gene region became popular approach of gene therapy. in detail, they could cleave multiple sites of mrna which are troublesome for the target to get repaired. silencing different crucial gene regions at a same time was expected to prevent the emergence of resistant viruses by lowering the possibilities of multiple mutation occurrences [ ] . the effects of sirnas could be enhanced by producing combinations pattern which can open a new window on gene therapeutics field. therefore, the primary aim of this study is to investigate the suitability of combination of sirnas acting as anti-cmv therapy in a proof of concept study. although sirna possess many advantages, unfortunately, the issue of sirna's off target effects still remain to be addressed and to deal with. cytotoxicity assay was performed to rule out any combinations that having detrimental effects on the ref cell viability. this is because introducing foreign particles could elevate the level of cytotoxicity of cells which may end up in cell death. unhealthy or dead cells are not able to sustain the virus production thus, can affects the results of sirna effectivity [ ] . therefore, before the commencement of combinations sirna effectivity assay, the cytotoxicity levels of the combinations were determined. the results displayed that these combinations sirna except dpb + dpc + ie b do not seem to induce a significant cytotoxicity level (p < . ) even at high concentration ( pmol) in different time points. however, the cell viability for all the sir-nas was found to be constantly decreasing slightly over time. two sirna combinations (dpb + dpc, dpb + ie b and dpc + ie b) with each pmol were found to be safe where cell viability was more than % at h at high concentration (final pmol). as expected, lower concentration starts from pmol (each . pmol) to pmol (each . pmol) was observed to have more than % of cell viability at , and h. however, quite high yet tolerable cytotoxicity level of three sirna combination (dpb + dpc + ie b) with each pmol was encountered with cell viability of % at pmol. since % of cytotoxicity level is within the tolerable range, the three-sirna combination (dpb + dpc + ie b) was included in the subsequent sirna experiments. in the present study, combination of sirnas targeting ie and dna poly gene regions were proven to suppressing the viral gene expression and inhibiting the replication of virus. the four combinations of sirna targeting same or different target regions of rcmv all- displayed effective inhibition of viral replication compared to scrambled sirna, negative control non-targeting sirna sample. compared to gcv, a positive control of treatment group, combinations of duplex sirnas: dpb + dpc, dpb + ie b and dpc + ie b showed better viral fig. alignment of dna sequences from combiantion sirnas treated group with stock virus for mutation identification. the sirnas treated rcmv all- dna was extracted and subjected for sanger sequencing. the obtained results were aligned using online clustal omega software. the synthesized sirna sequences was highlighted in blue. a dpb + dpc, b dpb + ie b, c dpc + ie b, d dpb + dpc + ie b inhibition on day and . however, combination of triple sirnas: dpb + dpc + ie b displayed lesser effective on controlling the viral replication compared to other group of treatments. the obtained results are in agreement with the relative quantification of gene expression by qpcr and also the rate of cpe formation. droplet digital pcr was employed to quantify the virus particles present in the treatment groups. although it has the same role as real-time pcr, but this novel technology is a splendid adaptation of the current pcr being more convenient to be used as it is highly precise and sensitive, hence making results more reliable and easier to reproduce [ , ] . more importantly, standard curve is not needed, and the results are interpreted directly by quantifying the droplets having the viral dna particles. the quantification results revealed that least number of virus particles was observed for dpb + dpc treatment group followed by dpb + dpc, and dpb + dpc + ie b, however, higher number was observed for dpc + ie treatment group. about % of viral dna copies reduction was observed for dpb + dpc and dpb + ie b while % for dpb + dpc + ie b treatment group. sadly, only % of viral dna copies were reduced for dpc + ie b presenting the least effective treatment group. from the detailed analysis, combination of sirnas having dpb sirna was found to perform better on virus inhibition rate compared to dpc and ie b. the obtained results were proving the capability of dpb sirna on inhibiting viral replication, which established as best sirna candidate compared to others on individual assessment section previously. except dpc + ie b, other combination of sir-nas portrayed better inhibition on rcmv all- dna compared to commercial drug gcv, which served as a positive control for treatment group. thus, this justifies the need of alternate gene therapy treatment to control the cmv disease more effectively. naturally, hosts have many mechanisms to alert their immune system and activating the pathways to block the production of new progeny virus from spreading to other uninfected cells upon infection. to serve the purpose, apoptosis, is an innate cellular defense mechanism responsible to promote programmed cell death, consequently preventing the persistent infection. apoptosis analysis revealed that combination sirnas treated group shows a good number of early apoptosis compared to late apoptosis and necrosis cells of untreated control group. out of four combinations of sirnas, dpb + dpc have significant number of viable and early apoptotic cells compared to late apoptotic cells. targeting dna polymerase, dpb + dpc combination has successfully inhibiting the virus replication, thus blocking its capacity to suppress the apoptosis pathway. on the other hand, the other three combination sirnas groups have similar pattern of results where most of the cells are accumulated on early apoptotic cells. no cells were identified on necrotic phase. as expected, majority of cells were observed under late apoptotic phase for negative control sirna and untreated groups. therefore, the combinations treatment proving the effectivity to suppresses the growth of rcmv all- which indirectly suppresses the apoptosis blocking pathway [ , ] . probably, combination sirnas have good synergetic effect on maintaining the health of host cells. few observations were noticed where combination of sirnas containing ie b showed reduce level of ie gene expression compared to dna poly. on the other hand, sirnas containing dpb and dpc have better mrna reduction level of dna poly gene expression compared to ie . for instance, dpb + dpc have high knockdown efficiency on dna poly gene ( %) compared to ie ( %) since both candidates are targeting the same gene region. our combination of sirnas treatment are in agreement with the findings of individual sirna where sirnas targeting dna poly gene region showed good reduction of mrna level especially dpb and dpc sirnas. next, combination of triple sirnas: dpb + dpc + ie b showed much lower mrna level ( - %) reduction for both ie and dna poly regions. this clearly showed that duplex sirna combinations portrayed better inhibition rate of gene expression compared to triple sirnas combination. the concept of more sirnas targeting different regions could effectively inhibit virus replication and gene expression seems to be unworkable in our experiments targeting rcmv all- . replication of virus in cell culture can be identified from distinctive morphologic changes of ref cell line designated as cytopathic effect also known as cpe [ ] . cytopathic action is commonly to giving us a clue whether the infectivity of virus is related to synthesis of infectious or noninfectious of virus particles. thus, this can be a preliminary indicator for the rcmv all- identification purpose. the cpe analysis was carried out on day pi. this is because approximately, it takes to days for cpe to starts and % cpe can be observed by day , as an agreement with cmv cell culture observation loh et al. [ ] . lesser cpe rate was observed for dpb + dpc sirnas treated group. the cells were started to enlarge, called as ballooning, which are very much better than the cpe conditions of gcv, negative control and untreated groups. next, dpb + ie b and dpc + ie b combinations were displayed similar pattern of cpe with gcv control group, where plaques can be seen clearly due to the de-attachment of the ref cells caused by rcmv all- growth progression. nevertheless, dpb + dpc + ie b combinations showed to be less prominent to inhibit viral cpe rate where majority of the cells were infected, and the plaques are expanding bigger. surprisingly, the cpe rate was about similar with untreated control group. achieving an effective antiviral therapy is much depended on low probability of drug resistance mutant development by an organism [ ] . to identify the effects of custom designed combination of sirnas effects on rcmv all- genome, the targeted region was sequenced and compared with stock virus. remarkably, at round of multiple sirnas treatment, no any significant mutation was observed compared to original stock. this experiment does convey an important message regarding the importance of sirna design approach. specific sirnas targeting highly conserved regions are capable to prevent or reduce the occurrence of mutant which can turn off the inhibitory effect of introduced sirnas. besides that, rcmv all- replication have been reduced by combination sirnas treatment which indirectly limiting the likelihood of mutation occurrence by inhibiting the growth of virus population carrying the mutated gene for infection. in addition, cmv is double stranded dna virus where the rate of mutation occurrence is low compared to other rna viruses. therefore, future studies on mutation identification need to be carried out at higher multiple round of sirnas treatment. in terms of performance, combination sirnas containing ie b especially dpb + ie b and dpc + ie b combinations displayed less efficient than dpb + dpc on inhibiting rcmv all- replication and gene expression. ie is the first ever gene to be transcribed after cmv infection. the ie products generated by differential splicing subsequently, will transactivate early genes. initially, it was expected that silencing ie gene region particularly ie could stop the activation of other genes. unfortunately, different scenario was observed where minimal expression of ie may adequate to turn on other genes, hence, progressing towards viral replication. this statement was supported by qpcr where high percentage of ie knockdown sirnas yet expressing dna polymerase and producing viral progeny. overall, according to the results obtained, combination of sirnas found to possess antiviral activity against rcmv all- , however, less impressive compared to highly efficient individual sirnas when applied at same total concentration at pmol. unfortunately, the synergistic effects of combination sirnas could not compete with the effectiveness of individual sirnas from all the gathered results of virus titration, virus particles quantification, mrna level investigation, cpe rate analysis and status of cell viabilities. the exact answer for the obtained results are unclear however, there are few reasons to explain the situation. first, there are higher chances for the sirnas to compete each other for the association with risc protein, which might have negative effects on each other's synergistic activity [ ] . second, the proportion of each sirna to get delivered into the cells is yet unclear even though the sirnas were mixed at equal concentration. may one can get transported into the cells more often compared to other which could lead for the failure of synergistic effects. next, the properties of sir-nas are varying among one another due to the different sequence composition, gc content, stability and affinity to bind the risc protein [ ] . more successful binding can direct the risc to target so that more frequent cleavage to occur at particular target. on the other hand, least successful sirnas binding to risc would have lesser cleavage frequency in turn having lesser effectiveness. thus, the individual characteristics of sirna could be superior to another which the stronger sirna have more silencing effects compared to weaker one [ ] . some studies have tried combination sirnas for the purpose of silencing viral gene target and cellular host factor which support the virus replication. for example, another study has been conducted and proved that hcv replication was inhibited pronouncedly by introducing sirnas which are targeting rna dependent rna polymerase viral gene as well la autoantigen of cellular factor [ ] . therefore, similar perspective of experiment can be conducted for rcmv all- in future to maximize the efficiency of combination sirnas on inhibiting the virus replication and gene expression. however, one needs to be more careful on choosing the cellular factor where the effects of silencing don't bring any detrimental harm for the host [ ] . taken all these together, combinations sir-nas application found to be beneficial in inhibiting the virus replication and more research need to be conducted to enhance the efficiency. despite human viruses, this system can be utilized for animal viruses which having an unbearable impact on poultry industries due to virus infections [ , ] . apart from that, this combinatorial sirnas approach could replace as an alternate treatment especially for plant-based vaccines which encountering difficulties during production [ ] . this is the first study initiated to investigate the effectiveness of sirna combinations to inhibit malaysian isolate cmv. the combinations were formulated based on the individual performance on inhibiting rcmv all- . the results markedly inhibited by viral genomic dna, gene expression and cmv replication in vitro, however, the findings are varying according to the combinations. the sirna combinations containing dpb sirna showed better rate of viral inhibition, as similar with the individual characteristics. overall, combinations having two sirnas showed promising results that the triple sirna cytomegalovirus replication steps and the actions of antiviral drugs the "silent" global burden of congenital cytomegalovirus cytomegalovirus (cmv) seroprevalence in the adult population of germany human herpes viruses in burn patients: a systematic review cytomegalovirus infection in immunocompetent and immunocompromised individuals-a review congenital and perinatal cytomegalovirus infection potent and specific genetic interference by double-stranded rna in caenorhabditis elegans molecular mechanisms and biological functions of sirna fundamentals of sirna and mirna therapeutics and a review of targeted nanoparticle delivery systems in breast cancer sirna for influenza therapy rna interference as a prospective tool for the control of human viral infections cholesterol modification 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replication combination of small interfering rnas mediates greater suppression on hepatitis b virus cccdna in hepg . . cells anti-hbv efficacy of combined sirnas targeting viral gene and heat shock cognate inhibition of vaccinia virus replication by two small interfering rnas targeting b r and g l genes and their synergistic combination with cidofovir a simple and cost-effective method for producing small interfering rnas with high efficacy synthesis and gene silencing properties of sirnas containing terminal amide linkages antisense molecules: a new class of drugs sirna therapeutics: a clinical reality clinical advances of sirna therapeutics the current state and future directions of rnaibased therapeutics droplet digital pcr versus qpcr for gene expression analysis with low abundant targets: from variable nonsense to publication quality data dpcr: a technology review viruses and apoptosis cytomegaloviruses inhibit bak-and bax-mediated apoptosis with two separate viral proteins chapter -antiviral therapy competition for risc binding predicts in vitro potency of sirna sirna efficiency: structure or sequence-that is the question predicting sirna efficacy based on multiple selective sirna representations and their combination at score level rna interference-mediated targeting of human cytomegalovirus immediate-early or early gene products inhibits viral replication with differential effects on cellular functions an association of orf virus infection among sheep and goats with herd health programme in terengganu state, eastern region of the peninsular malaysia plant-based vaccines: production and challenges publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. combination. interestingly, no any mutation occurrence was encountered among the treated groups thus highlighting its feasibility to be used as treatment option in future. supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file : figure s . standard curve and amplification curve of the sirnas and gapdh were determined from tenfold serial dilution of rna isolated from treated cells. figure s . the cytotoxicity of combinations sirna targeting rcmv all- and negative control sirna in ref cells. cells were transfected with lipofectamine and sirna complex, which were prepared in final concentration of pmol of sirna and incubated for h and an additional of / / h at ℃. the cellular viability was measured by mtt assay. each treatment was performed in triplicate and repeated in two independent experiments. results represent mean ± standard deviation. figure s . gating references used for unstained, pi and fitc apoptosis analysis using flow cytometry. figure s . d ddpcr analysis plot shows detection of rcmv all- dna in sirna treated and non-treated groups. each sample was partitioned into an average of , droplets per well and replicated in two wells. the droplet counts for positive (blue) and negative (gray) from all replicated wells were combined to yield a "merged" well. a fluorescence amplitude value > (pink line) was considered to be a positive droplet. the authors were hereby given a declaration that this work was done by all of them named in this paper and all liabilities pertaining to claims relating to the content of this article will be borne by them. knb, aaa and jab conceived the idea, participates in data collection and run the test. caca, ffaj, mmn and mlma participated in conceptualization of the idea, study design, consultation, review, and editing of paper, knb, caca, ffaj, mmn and mlma involved in results interpretation as well as preparation of the final manuscript draft. all authors read and approved the final manuscript. this study was funded by ministry of higher education malaysia vot no: and ips universiti putra malaysia vot no.: . all data generated or analyzed during this study are included in this manuscript. the study does not contain any experiments involving human or animal participation, and all experiments in this study are in compliance with ethical standards for research. not applicable. the authors declare that they have no any conflicts of interest regarding the key: cord- -jh omg authors: nobach, daniel; herden, christiane title: no evidence for european bats serving as reservoir for borna disease virus or other known mammalian orthobornaviruses date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: jh omg background: the majority of emerging infectious diseases are zoonotic in nature and originate from wildlife reservoirs. borna disease, caused by borna disease virus (bodv- ), is an infectious disease affecting mammals, but recently it has also been shown to cause fatal encephalitis in humans. the endemic character of borna disease points towards a nature-bound reservoir, with only one shrew species identified as reservoir host to date. bats have been identified as reservoirs of a variety of zoonotic infectious agents. endogenous borna-like elements in the genome of certain bat species additionally point towards co-evolution of bats with bornaviruses and therefore raise the question whether bats could serve as a potential reservoir of orthobornaviruses. methods: frozen brain samples (n = ) of bats of seven different genera from germany were investigated by orthobornaviral rt-pcr. additionally, tissue slides of formalin-fixed paraffin-embedded material of a subset of these bats (n = ) were investigated for orthobornaviral phosphoprotein by immunohistochemistry. results: the brain samples were tested by rt-pcr without any evidence of orthobornavirus specific amplicons. immunohistochemistry revealed a faint immunoreaction in / bats but with an untypical staining pattern for viral antigen. conclusions: rt-pcr-screening showed no evidence for orthobornaviral rna in the investigated bats. however, immunohistochemistry results should be investigated further to elucidate whether the reaction might be associated with expressed endogenous bornaviral elements or other so far unknown bornaviruses. the increasing incidence of emerging infectious diseases (eid) represents a threat to public health. interestingly, the majority of these eids are zoonotic in nature and originate from wildlife reservoirs. due to their biological characteristics, particularly bats have been identified as reservoirs for many emerging viruses [ ] . many of these emerging viruses are rna-viruses of the order mononegavirales. in this order, the virus family bornaviridae has been growing remarkably during recent years due to the discovery of several new species and genera. as of , the taxonomy comprises three genera: carbovirus, cultervirus and orthobornavirus [ ] . of the genus orthobornavirus, two species, mammalian orthobornavirus and mammalian orthobornavirus, are known to affect mammals. belonging to mammalian orthobornavirus, borna disease virus (bodv- ) is still the most prevalent bornavirus in mammals. bodv- is well known to cause severe and fatal neurological borna disease in a variety of mammals, mainly horses and sheep. recently bodv- was identified as causal agent in fatal human encephalitis cases [ , ] . this underlines the need to unravel potential sources of infection in order to prevent further animal and human cases, especially since no curative therapy or vaccination exists [ , ] . endemic areas for borna disease are located in central europe, such as bavaria, saxony-anhalt and saxony in germany, st. gallen and canton of grisons in switzerland and vorarlberg and upper austria in austria [ , ] . phylogeny of bodv- isolates reflects their geographical origin and respective endemic regions regardless of the host species they have been isolated from [ ] . the endemic character, strong conservation of the viral genome and seasonal occurrence of the disease already pointed to a potential wildlife reservoir [ ] . as the sequences of bd cases from neighbouring locations are particularly stable even over years, this wildlife reservoir was assumed to be territorially bound [ ] . in several of the endemic areas, the bicolored white-toothed shrew (crocidura leucodon), belonging to the order of eulipotyphla, has been identified as natural reservoir of bodv- [ , , ] . however, beside the bicolored white-toothed shrew, no other reservoir, neither eulipotyphla nor rodent, has been found yet. bank voles have been experimentally proven to be susceptible to bodv- [ ] and serum antibodies against bornaviruses have been detected in free ranging bank voles [ ] . nevertheless, there is no evidence for naturally infected bank voles in endemic areas to date. serological data have shown that several other free ranging small mammals, mostly belonging to the order of rodentia, can also exhibit serum antibodies against bornaviridae, but without any other evidence of bodv- -infection [ ] . only bicolored white-toothed shrews display a disseminated virus distribution and harbour bodv- in excretory and secretory organs [ , ] and shed infectious virus which suggests that they can transmit bodv- [ ] . although several rodents and other small mammals are known as important reservoirs for many viruses, bats (order: chiroptera) represent the vast majority of identified natural reservoirs of several virus families/species to date [ , ] . for example, bats are known reservoirs for a growing list of rna viruses, including rabies virus and other lyssaviruses, henipaviruses, coronaviruses and ebola virus [ ] . virus infections in bats follow the typical pattern of reservoirs with a persistent course lacking clinical disease [ , ] . several biological characteristics including gregariousness with large colonies, seasonal migrating and long life span make bats suitable to carry and spread viruses [ ] . seasonal migrating and wide hunting territories of bats can lead to wide distribution of harboured viruses [ ] , however, shedding in pulses and additional local factors can lead to local transmission events [ ] . in europe, bat species from the order chiroptera can be found [ ] . these european bats share the same biological characteristics, but transmission events of zoonotic viruses are rare due to smaller dimensions of populations and colonies [ ] . in germany, bat species can be encountered from the family rhinolophidae and vespertilionidae [ ] . they include common native species, such as members of the genera pipistrellus, myotis and vespertilio, and some seriously endangered species like rhinolophus hipposideros, myotis emarginatus and barbastella barbastellus [ ] . several bat species, for example myotis natteri, use roosts in barns and stables [ ] , which facilitates the possibility of virus transmission to livestock [ ] . in addition, endogenous bornavirus-like elements (ebl) have been detected in the genome of several bat species [ ] [ ] [ ] [ ] . these ebl are dna-sequences in bat genomes displaying considerable sequence identities to present-day bornaviral genes on amino acid level of about - % [ ] . in some bat species, transcription of ebl similar to the bornaviral rna-dependent rnapolymerase has been reported [ ] . further analysis of these ebl strongly hint at ancestral and repeated contact between bats and bornaviruses during their evolution at least , million years ago [ , ] . the function of these ebl is still discussed and an immunological benefit in the interaction with bornaviruses has been suggested in some mammalian species [ , ] . as the retaining of the expressed ebl in the bat genome despite evolutionary selection requires resources, a benefit of the ebl and regular encounters between bornaviruses and bats during evolution seem plausible [ ] . in conclusion, due to the continuous detection of new viruses in bats, the unclear situation regarding additional potential bodv- -reservoirs and molecular evidence for co-evolution of bats and bornaviruses, this study was conducted to investigate the potential presence of the most common orthobornaviruses in bats from endemic and non-endemic areas in germany. two hundred fifty-seven brain samples of bats from germany ( from endemic regions in bavaria) were provided by the leibniz institute for zoo and wildlife research (leibniz-izw), berlin ( pipistrellus sp., nyctalus sp., myotis sp., eptesicus sp., vespertilio sp., plecotus sp., barbastella sp., bat without species identification), and stored frozen at − °c. additionally, bat organs from diagnostic necropsies archived as formalin-fixed paraffin-embedded (ffpe) material of bats from the german federal state of bavaria ( pipistrellus sp., vespertilio sp., eptesicus sp., nyctalus sp., plecotus sp., myotes sp.) and bats from the federal state of hesse ( myotis sp., pipistrellus sp., plecotus sp., nyctalus sp., bats without species identification) were provided by the leibniz-izw, the state veterinary institute of giessen (landesbetrieb hessisches landeslabor) and the department of animal ecology and systematics, giessen. all organ tissues were retrieved from diagnostic necropsy material from carcasses submitted by bat rehabilitation centres and bat researchers in germany to the respective institution. samples from the leibniz-izw were archived materials from a previous larger study on disease and causes of death in european bats from germany [ ] . for screening for orthobornaviral rna ( bp of x/ p-orf), brain samples were analysed by a two-step rt-pcr detecting a broad spectrum of orthobornaviruses (see below). rna isolation and rt-reaction was performed with rneasy mini kit (qiagen) and quantitect reverse transcription kit (qiagen) according to manufacturer's instructions, respectively. pcr was performed with mytaq hsmix (bioline) under manufacturer's standard condition with degenerated primers (additional file : table s ) [ ] . these primers were designed to detect viruses of seven species of the genus orthobornavirus (mammalian orthobornavirus, mammalian orthobornavirus, passeriform orthobornavirus, passeriform orthobornavirus, psittaciform orthobornavirus, psittaciform orthobornavirus and waterbird orthobornavirus), but not viruses of the species elapid orthobornavirus of the genus orthobornavirus or viruses of the genera carbovirus or cultervirus. the applied rt-pcr assay has been proven to detect several known pathogenic members of the genus orthobornavirus (bodv- , variegated squirrel bornavirus (vsbv- ), parrot bornavirus (pabv- ), parrot bornavirus (pabv- )) [ ] . as internal control, glyceraldehyde- phosphate-dehydrogenase-(gapdh)-amplification ( bp) was included. as positive control, isolated rna from a bodv- -positive mouse was used, and a formerly negatively tested bat served as negative control. lengths of amplicons were visualized with gel electrophoresis ( % agarose gel with % midori green (biozym)) according to manufacturer's instructions and commercial sanger sequencing of orthobornaviral amplicons was performed for positive controls (gatc, eurofins genomics). bodv- negatively-tested bat-rna was spiked with serial dilutions of either bodv- -rna, vsbv- -rna, pabv- -rna or pabv- -rna to assess specificity and sensitivity. to screen for bornaviral antigen, immunohistochemistry was performed using a polyclonal antibody for the detection of bornaviral phosphoprotein (antibody p ). this antibody is known for its cross-reactivity also with the phosphoprotein of pabv- and pabv- of the species psittaciform orthobornaviruses [ ] and vsbv- [ ] . all reactions were compared to a negative control slide incubated with a rabbit serum (rabbit immunoglobulin fraction, dako). organs with positive immunostaining were further examined with a panel of antibodies to examine specificity of this reaction. the panel included two antibodies directed against the viral nucleoprotein of bodv- (monoclonal antibody bo [ ] and polyclonal antibody anti-bodv-n [ ] ) and a mix of polyclonal antibodies detecting vsbv- nucleoprotein and phosphoprotein [provided by dennis tappe, bernhard nocht institute hamburg]. to exclude unspecific reaction of the polyclonal rabbit-antibodies, a polyclonal antibody detecting rabies virus as well as a second control rabbit serum (thermofisher) were used as additional negative controls (details on immunohistochemistry protocols in additional file : table s ). by rt-pcr-screening, in / samples gapdhamplicons could be obtained, the other samples were excluded due to insufficient quality. these samples were tested for orthobornaviral rna and no specific amplicons regardless of origin from endemic or nonendemic areas were observed. the control consisting of rna of a bodv- infected mouse was correctly amplified as verified by correct size on the gel and respective sequences (additional file : figure s ). spiking of bat rna with serial dilutions of various orthobornavirus-rna demonstrated the detection limit of orthobornavirus copies in ng rna. by immunohistochemistry applying the polyclonal antibody p specific for the phosphoprotein, a faint reaction was found in / animals, in particular located in the cytoplasm of smooth muscle cells of the intestine. all respective negative control slides were without any immunoreaction regardless which control antibody was used. no immunoreactivity was found using the monoclonal antibody bo specific for the bodv- nucleoprotein in these samples. immunoreactivity was found using the polyclonal antibody detecting bodv- nucleoprotein in one sample and using the polyclonal antibodies detecting vsbv-nucleoprotein and phosphoprotein in two samples. however, in / other animals a comparable immunoreaction was observed in negative control slides using control rabbit serum or polyclonal anti-rabies antibodies (details in additional file : table s and additional file : figure s ). recent cases of fatal encephalitis in humans due to bodv- infection strengthen the need to survey potential wildlife reservoirs and identify potential risk factors for infection. although the bicolored white-toothed shrew has been identified as indigenous reservoir of bodv- , other potential reservoirs or animal carriers are still unknown so that further investigations of small mammals including bat species are urgently needed. bats have already been discovered as reservoir of emerging and highly pathogenic viruses. many factors, such as their gregarious way of life, can facilitate pathogen transmission to other bats and virus persistence in the population. in european bats, only few zoonotic viruses have been discovered [ , , ] and the overall hazard for humans is comparably low [ ] . as some bats take roosts in barns and stables [ ] and bat carcasses are found in close proximity to agriculture [ ] , a risk of sporadic transmission events to livestock animals can be assumed if viruses can be detected. animal movement across borders of endemic regions during hunting and migration of bats seems to contradict stable geographical clustering of bodv- isolates and the hypothesis of a territorially bound reservoir [ ] . nonetheless, consistent usage of the same roosts as summer or winter quarter may support observed clustering and could facilitate rare endemic transmission. additionally, the molecular evidence for co-evolution of bats and bornaviruses [ , ] could suggest the possibility of infections of other potentially so far unknown bornaviruses beside the tested orthobornaviruses. therefore, this study aimed to examine the possible role of bats as carrier and reservoir of orthobornaviruses such as bodv- as one of the most common virus. all samples originated from bats which died because of injuries or disease. they were part of a previous study on diseases and causes of death in european bats, where traumatic injuries and inflammatory lesions, partly due to bacterial infections, were the major cause of deaths in these animals [ ] . since borna disease in animals is known to be endemic in specific areas of germany [ ] , samples were sorted by regional origin corresponding to known endemic regions (bavaria) and other nonendemic regions in germany. however, as some bat species tend to have wide hunting territories or migrate during the year and can cross the borders of endemic regions, this sorting might bear a risk of bias. the study includes samples from available bat species and is not limited to bat species suspected to interact with bornaviruses [ , ] as interspecies virus transmission has already been observed [ , ] . interestingly, the screening by rt-pcr for orthobornaviral rna provided no evidence of orthobornavirus infection in the investigated bats. the detection limit of the applied orthobornavirus rt-pcr was . ng/μl orthobornaviral rna in ng/μl mammalian rna according to literature [ ] and copies in ng rna in our own testing. therefore, already low amounts of viral rna should have been detected as verified by the correct amplification of all control orthobornavirus species. spillover host [ ] and reservoir species [ ] regularly yield high amount of viral rna, much higher than the detection level of the applied rt-pcr assay. however, the presence of previously undiscovered bornavirus species, such as the ones recently described in reptiles and classified as carboviruses [ ] cannot be excluded and could be further investigated by broad and undirected approaches, such as metagenomics. in contrast to the rt-pcr results, the faint immunohistochemical reaction in smooth muscles of three animals raises the question whether an antigen with cross-reactivity or a bornaviral phosphoprotein is present. however, in several other animals similar reactions were detected applying unrelated polyclonal antibodies produced in rabbits and unspecific rabbit serum. moreover, the staining pattern is rather untypical for bodv- but occurs regularly in avian bornavirus infections [ ] . the immunohistochemical reaction was not observed using a monoclonal antibody against the nucleoprotein of bodv- . it was observed in one animal using a polyclonal antibody against the nucleoprotein of bodv- and in two animals using polyclonal antibodies against proteins of variegated squirrel bornavirus- . thus, an infection with another, so far unknown bornavirus could not completely be excluded and has to be investigated in further studies. scarcity and limited quality of material impeded further immunohistochemical and molecular investigations. as endogenous bornaviral elements similar to bornaviral rna-dependent rnapolymerase have been found in bats [ ] , a translated endogenous element could also have been detected by immunohistochemistry due to cross-reactivity. however, an endogenous bornaviral-phosphoprotein-like protein has not been found yet and some authors have discussed a deleterious effect of endogenization of bornaviral phosphoprotein [ ] . on the contrary, an endogenous bornaviral-p-like protein might also help prevent against bornavirus infection as the bornaviral polymerase is inhibited by a disturbed nucleoprotein-phosphoproteinration [ ] . to summarize, rt-pcr-screening of tissues from european bats revealed no evidence for orthobornaviral rna. further studies could unravel whether the immunohistochemical reactions might be due to expression of endogenous sequences gained during evolution of the bat species or even new bornaviruses. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : figure s . gel electrophoresis of pcr products. - : borna-negative samples of sufficient quality with gapdh-band at bpamplicon length; : sample of insufficient quality without gapdh-band; ntrt: no template reverse transcription-reaction control; pc: positive control (bodv- -positive mouse); nc: negative control (bodv- negative bat); ntc: no template control of pcr; bp: base pairs. additional file : table s . primer sequences. information about primers. 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quantification of borna disease virus in diseased hosts divergent bornaviruses from australian carpet pythons with neurological disease date the origin of extant bornaviridae prior to the end-cretaceous extinction paleovirology of bornaviruses: what can be learned from molecular fossils of bornaviruses active borna disease virus polymerase complex requires a distinct nucleoprotein-tophosphoprotein ratio but no viral x protein publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors want to thank silke engel for help with the rt-pcr, silke gantz and diana waldschmidt for help with the immunohistochemistry and jana müller for fruitful discussions during writing and proofreading. the authors are grateful to marion biering, kristin mühldorfer and gudrun wibbelt, leibniz institute for zoo and wildlife research, berlin, jorge a. encarnação, department of animal ecology and systematics, and anne nesseler, state veterinary institute of giessen, for sharing their archival samples. authors' contributions dn contributed to the conception of the work, performed the laboratory work (rt-pcr, ihc) and analysed the data and wrote the manuscript. ch contributed to the conception of the work, the acquiring of samples and the interpretation of data and manuscript writing. all authors read and approved the final manuscript. the authors declare that they have not received any funding. all data generated or analysed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate all work was done according to the respective laws of the federal republic of germany. no additional ethic approval was required. not applicable. the authors declare that they have no competing interests. key: cord- -tkvfneje authors: mendez, jairo a; usme-ciro, jose a; domingo, cristina; rey, gloria j; sanchez, juan a; tenorio, antonio; gallego-gomez, juan c title: phylogenetic history demonstrates two different lineages of dengue type virus in colombia date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: tkvfneje background: dengue fever is one of the most important viral re-emergent diseases affecting about million people around the world especially in tropical and sub-tropical countries. in colombia, the virus was first detected in the earliest 's when the disease became a major public health concern. since then, all four serotypes of the virus have been reported. although most of the huge outbreaks reported in this country have involved dengue virus serotype (denv- ), there are not studies about its origin, genetic diversity and distribution. results: we used bp corresponding to the carboxyl terminus of envelope (e) gene from colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. analyzed denv- colombian isolates belonged to the formerly defined genotype v. only one virus isolate was clasified in the genotype i, likely representing a sole introduction that did not spread. the oldest strains were closely related to those detected for the first time in america in from the caribbean and were detected for two years until their disappearance about six years later. around , a split up generated lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. conclusion: denv- has been circulating since in colombia. yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype v, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. we report the rapid spread patterns and high evolution rate of the different denv- lineages. dengue virus infection has been an important impact on humans over the last several years, with an estimated million dengue infections and an average of million cases reported annually in more than countries in tropical and subtropical regions [ ] [ ] [ ] [ ] [ ] . this mosquitoborne flavivirus causes a wide spectrum of clinical manifestations in humans, which include an acute self-limited flu-like illness known as dengue fever (df). df is characterized by headache, myalgia, arthralgia, retro-orbital pain and sometimes maculopapular rash. dengue haemorrhagic fever (dhf) is a severe illness documented by haemoconcentration (haematocrit increase by %) and evidence of plasma leakage such as pleural effusion and ascites as the major pathophysiological features. in some patients, dhf may progress to hypovolemic shock (dengue shock syndrome, dss) with circulatory failure [ , [ ] [ ] [ ] . dengue virus (denv) is an enveloped virus with a positive sense ssrna of about kb coding a single open reading frame for three structural proteins, core (c), premembrane/membrane (prm/m) and envelope (e), and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, ns ). based on serological analysis, denv can be differentiated as four distinct serotypes (denv- , denv- , denv- and denv- ), each one with the capacity to infect and cause even the more severe manifestation, although some serotypes have been isolated more frequently in dhf epidemics. on the other hand, evolution studies and molecular epidemiology using nucleotide sequences from the denv genome have demonstrated the occurrence of genotype clades within each serotype [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, genetic characterization of denv has become a critical issue for understanding epidemic patterns of viral spread. at the same time, the important role of denv itself in disease severity has also been proposed rather than the immune enhancement developed after subsequent infection with heterologous serotypes [ , , ] . the increase in virus transmission over the last years has possibly increased its adaptive potential. in addition, host factors such as the age, race, presence of non-neutralazing cross-reactive antibodies and possibly chronic diseases could act as selective pressures, resulting in more virulent genotypes that may be associated with dhf/dss [ , , [ ] [ ] [ ] [ ] [ ] [ ] . four denv serotypes have been involved in colombian epidemics, although denv- and denv- have the higher circulation rate since [ , , ] . moreover, since the first case of dhf in colombia at the end of , these two serotypes have been associated with severe disease. to date, denv- falls into five clades designated as genotype i (southeast asia, china and east africa), genotype ii (thailand), genotype iii (malaysia), genotype iv (south pacific) and genotype v (america, africa). additionally, the existence of lineages with distinctive geographical and temporal relationships had been suggested [ , , , , [ ] [ ] [ ] [ ] . due to the importance of denv in public health, the particular goals of this research were to reconstruct the phylogenetic history of denv- and to date the phylogenetic tree using isolation time as calibration points to establish date of introduction of virus and rate evolution patterns of virus in colombia. seventy four viruses obtained from symptomatic patients were isolated in mosquito cell culture and subsequently identified as denv- serotype by monoclonal antibodies and confirmed by rt-pcr methods. from the samples, it was not possible to obtain the exact geographic origin of samples. the remaining isolates are listed in table indicating locality, isolation year, accession number and genotype. sequences from the carboxyl terminus of the envelope (e) gene from the colombian denv- isolates were aligned in clustal w [ , ] and compared with previously reported sequences elsewhere, resulting in a trivial alignment as long as there were no indels in the sequences alignment. the maximum likelihood analysis comparing sequences is presented in figure . previously reported genotypes were represented in the tree and placed most of the colombian isolates nesting in the genotype v clade (america, africa) and were closely related to argentina, brazil and paraguay virus strains. nevertheless, the oldest sequences denv- /co/ _atlantico/ , denv- /co/ _choco/ and denv- /co/ _choco/ were slightly distant from the remaining strains and appeared in close proximity to some caribbean island and other american isolates (trinidad, french guinea). interestingly, the isolate denv- /co/ _valle/ appeared in a different clade, as the sister branch of japan (denv- /jp/mochizuki/ ), china (denv- / cn/gz- / ), ethiopia (denv- /dj/ethiopia/ ) and cambodia (denv- /kh/ ) strains, which have been defined as lineages of the genotype i [ ] . to allow a better resolution of the tree, we performed a phylogenetic reconstruction using only the colombian isolates. again, the oldest isolates were more divergent representing the first entrance of virus in colombia. although the genotype v was the only represented in the tree, two different lineages may be defined based on cladal distribution (figure ). we used a bayesian inference based on mcmc to reconstruct colombian denv coalescent history. beast allowed the use of isolation year as calibrating point to estimate divergence time and then generated a posterior probability (pp) distribution of trees instead of a bootstrap value [ ] [ ] [ ] [ ] . the resulting tree clearly placed the genotypes of denv- already circulating globally before the first appearance of this serotype in the americas between and (figure ). according to the % highest posterior density (hpd) beneath the strict clock model, the estimated root for this phylogeny was and the substitution rate was . × - substitutions per site, per year. to increase resolution, we use the strict molecular clock model to reconstruct colombian isolates ( figure ). under the assumption of a constant substitution rate, the estimated root indicates as the date of the more recent common ancestor. in addition, there was a split up around between denv- /co/ _guaviare/ isolates and the remaining strains (pp = , ). as the time goes by, we can see a sustained increase in number of isolates and a rapid spread of viruses, which included few changes among them as seen with the branch lengths. by the year (approximately), another remarkable partition event occurred to generate well defined clades (pp = , and ), evolving independently since the early ′s until recent time. emerging and re-emerging diseases have become a public health major concern in developing countries, where dengue is perhaps the most important vector-borne viral disease in terms of morbidity. in colombia, df and dhf had been associated to the four denv serotypes with denv- and denv- predominating since after the re-appearance and spread of aedes (stegomyia) aegipty [ ] . denv- circulated for a short time in and then it was not detected until when re-introduction occurred probably from venezuela [ ] . denv- has been detected sporadically every year since , when it was involved in several df cases. the huge genetic diversity of denv has been vastly documented, starting perhaps with the rico-hesse proposal of different "genotypes" comprising serotypes and [ ] , following by several studies and genotype definition of denv- and denv- . in this way, five different genotypes has been previously defined for denv- (genotypes i to v) suggesting a significant genetic variation. in fact, various lineages had been proposed based on time-spatial clustering and clade distribution [ , , [ ] [ ] [ ] [ ] . in the present study, colombian denv- sequences were analyzed to try to reconstruct the phylogenetic history of the virus in this country. different genome regions have been used to infer denv phylogeny including those with short fragments [ , , ] . here we employed a sequence from the carboxi terminal of the envelope (e) protein which has demonstrated to provide a useful phylogenetic signal to define genotype clustering [ ] . it is important to note that the better resolution of evolutionary patterns should be obtained from complete genomes. however, it was not possible to obtain largest fragments from the oldest isolates, probably because of rna degradation across the time. as expected, all strains were clustered with those from brazil, paraguay, argentina, and different caribbean islands, corresponding to the formerly named genotype v (america/africa), showing a well supported clade clearly separated from the others genotypes. colombian strains denv- /co/ _atlantico/ , denv- /co/ _choco/ and denv- /co/ _choco/ , were separated from the remaining isolates and appeared closer to those from the caribbean islands, which represent the entrance of serotype into the americas. it was reported for the first time in in jamaica and rapidly spreading to the antilles including cuba, antigua & barbuda, aruba, bahamas, barbados, curaçao, dominica, grenada, guadaloupe, guyana, haiti, martinique, montserrat, puerto rico, st. kitts, st. martin, st. vincent and the grenadines, trinidad, turks and caicos, and the virgin islands [ ] . in , denv- was implicated in large mainland outbreaks perhaps occurring at the same time in colombia, venezuela, surinam, french guyana, and eventually centro america and mexico. in colombia, denv- was isolated between and , so the strain denv- / co/ _atlantico/ represents perhaps the first virus entrance to the country. it rapidly spread until the next isolation in choco (denv- /co/ _choco/ and denv- /co/ _choco/ ) and then it fades away (or at less it was not reported) probably displaced by denv- (maintaining denv- in a silent low circulation) until when it established in different localities. it is important to note that even with the mobility between countries and increasing opportunity of viral introduction, only one denv- genotype is circulating in america, different to denv- and denv- of which at least genotypes has been detected (america/asia genotypes and i/iii genotypes respectively) suggesting phylogenetic analyses were conducted in paup* perhaps, dissimilar patterns of viral spread and transmission between denv genotypes and even different adaptation capacity. many researchers have categorized denv in non official taxonomic levels beneath genotype, based specially in cladal distribution or geographical clustering. circulation of these "lineages" has been particularly defined for denv- in india, where at least different lineages had been proposed (india- close to american strains, india- related to singapore isolate, india- in south india and india- from delhi and gwalior) [ , ] . in our study, a remarkable cladogenesis event occurs around according to the molecular clock, generating two well supported clades corresponding to putative colombian denv- lineages. despite the eco-epidemiology similarities between colombia and neighbor countries were dengue is a major concern, lineages have not been previously demonstrated for denv- . in fact, according to ml phylogeny, most of the american strains (argentina and brazil) correspond to the lineage- , leaving the lineage restricted to colombia. although geographic distribution of these lineages is not clearly delimitated, it is evident that they are evolving independently and most likely in parallel at the same localities. despite the emergence and rapid diversification of denv has been a matter of special concern, precise mechanisms of evolution remain unclear [ ] [ ] [ ] [ ] [ ] [ ] . it is a fact that human rna viruses including influenza, hiv, coronavirus, etc., have particularly increased mutation and evolution rates mostly because of the lack of proofreading activity of rna-dependent rna-polymerase [ , ] . nevertheless, arthropod-borne viruses (arboviruses) have demonstrated slower mutation rates comparing with those infecting directly human host, probably because of the trade-off effect occurring when the virus is obligated to adapt alternatively into the invertebrate vector and vertebrate host [ ] . this resulting constrain has been experimentally assessed in vivo to venezuelan equine encephalitis [ ] and in vitro to denv [ ] demonstrating that fitness improves when virus specialize in a single cell line but decreases in virus undergoing alternative passages in different cells. in view of that, over all denv mutation rates have been previously inferred, ranging from . × - (denv- ) to . × - (denv- ) [ ] . in the present study, we found a mutation rate of . × - substitutions per site, per year, suggesting faster evolution rates for colombian strains, perhaps because of the high transmition occurrence especially in hyperendemic areas, where virus replicates in several human hosts, reducing the constraining effect occurred in the vector. however, this high mutation rate does not necessarily reflect a fitness advantage or a successful adaptation process. actually, positive selection for denv seems to be serotype/genotype dependent and even more, protein specific. in fact, envelope (e) protein apparently exhibits some adaptation evidence in denv- , denv- and various denv- genotypes, but not for denv- , strongly suggesting a purifying selection pressure, at least over this gene. nevertheless, further studies have to be done to try to understand the adaptation process in denv. on the other hand, although mostly of colombian strains belong to the genotype v, there is an isolate, denv- /co/ _valle/ placed into genotype i near to asia, china and east africa strains. the ml tree show this strain close to denv- /jp/mochizuki/ , a strain considered extinct. since we do not have this virus as reference in our laboratory, we can discard cross contamination during the assay. moreover, the presence of this virus could be explained based on the migration process occurred from asia to america, officially starting to colombia by , and sustained until the mid xx century [ ] . thus, establishment of asian colonies increased visitors and perhaps favored the entrance of viral strains. we can speculate that those viruses did not fit to the new environment and the adaptation events were constrained because of the selective pressures including different vectors and human immune response. according to natural history of denv, evolution events could bring new genetic variants and eventually increase the severity of disease. although pathogenic markers remain unclear, hemorrhagic features on some asian denv- genotypes have been demonstrated and asian derived denv- genotypes associated to dengue fever and dengue hemorrhagic fever have been reported in brazil [ ] . moreover, changes in clinical manifestation of disease (atypical dengue) such as viscerotropism or encephalitis may respond to the circulation of new denv lineages with increased pathogenic potential. consequently, epidemiological programs should include not just virological diagnosis but genotype surveillance too. this study shows in a defined time-scale, not just the first entrance of denv in colombia, but also the viral evolution process in a highly endemic area. as a major conclusion, only one genotype of denv has been circulating since the first epidemic reports in the continental area. nevertheless, two different lineages have been evolving fast since the earliest ′s according to molecular clock. as these evolution events may derive in a marked pathogenic potential, surveillance programs should include molecular methodologies. in fact, unusual presentation of disease currently reported by local health care institutions may be correlated to this evolution process. further analyses by using at least complete e gene should be done to corroborate our results. denv- strains used in this study were obtained from the virus collection of the national health institute (ins, virology lab, bogotá, colombia), and comprise isolates from outbreaks, epidemics and routine epidemiological surveillance. clinical samples were collected between and from different localities all around the country, so they represent most viruses circulating in colombia during the last years (table ) . all viral stocks were inoculated on c / aedes albopictus cells growing in eagle's minimal essential medium (e-mem) supplemented with % fetal calf serum (fsc). after days of incubation at °c, monolayer was disrupted and supernatant was then recovered by centrifugation and stored at - °c until use. the remained cells were washed with phosfate buffer saline (pbs) and dripped on slides; after fixed in cold acetone, slides were incubated with monoclonal antibodies (anti-denv- to anti-denv- , kindly donated by cdc, puerto rico) for one hour, washed with pbs and incubated again with a fluorescent conjugated antibody. additionally, denv- serotype confirmation was done by reverse transcription polymerase chain reaction (rt-pcr) using specific primers [ ] . cell culture supernatants were used to extract viral rna using qiaamp viral rna minikit (qiagen, germany) following manufacturer's instructions. briefly, μl of each supernatant was placed into μl of avl buffer with . μl of carrier rna and mixed with ethanol ( - %) before passed through a column by centrifugation. after washing with buffers aw and aw rna was finally eluted with μl of ave buffer and stored at - °c until use. five microliters from each rna extraction were used as template in a one step rt-pcr reaction (qiagen, one-step rt-pcr kit) as previously described [ ] . primers used [den s ( ′-tggctgagacc-carcatggnac- ′) and den as ( ′-caatt-catttgatatttgyttccac- ′)] were designated to amplify bp from de joining region e/ns . reactions were evaluated in % agarose gel stained with ethidum bromide and reactions observed as negative were then subjected to nested pcr as follow: μl of initial rt-pcr product, × buffer b ( mm tris-hcl ph . , mm mgcl , mm (nh ) so ), pmol of each primer [den s ( ′-ggaaaatgttygaagcaacyg ccc- ′), den as ( ′-tcctcccatgccttcc-cratgg- ′)] and . u of taq dna polimerase (invotrogene) in a final volume of μl. pcr reactions were first denaturated at °c ( minutes) and then subjected to cycles of denaturation ( °c, seconds), primer annealing ( °c, minutes), primer extension ( °c, seconds) and a final extension step at °c for minutes. nested pcr was evaluated in % agarose gel stained with ethidium bromide. amplified products (from rt-pcr or nested pcr) were purified using qiaquick pcr purification kit (qiagen, germany) and then used as template for sequencing reactions using the abi prism dye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca). sequencing was carried out on both strands with pmol of primers used for nested pcr, and the products were analyzed using an abi model automated sequencer (applied biosystems, usa). overlapping sequences for each sample obtained from sense and antisense primers were combined to obtain a consensus sequence using the seq-man module of lasergene (dnastar inc. software, madison, wis.). a total of bp [corresponding to carboxyl terminus of envelope (e) gene] from new sequences were compared with previously sequenced strains from all over the world, available in genbank. consensus sequences were aligned using the program clustal w included in mega package version . [ , ] . phylogenetic trees were constructed with the maximum parsimony and maximum likelihood (ml) methods incorporated in the paup* . program [ ] . phylogenetic analyses were performed by using the best model of nucleotide substitution based on modeltest [ ] (analyses are available upon request). statistical significance of tree topology was assessed with a bootstrap with replicates. obtained trees were visualized using the tree view program [ ] . in addition, estimated rate of evolutionary change (nucleotide substitutions per site per year) and tree root age was obtained with the program beast (bayesian evolutionary analysis by sampling trees) [ ] , which uses bayesian markov chain montecarlo (mcmc) algorithms combined with the chosen model and prior knowledge of sequence data to infer the posterior probability distribution of phylogenies [ ] [ ] [ ] [ ] . we analyze the data using the year of isolation as calibration points to estimate divergence time in years. in order to avoid duplicates, sequences identical to other on the dataset were removed. rate variation among branches was inferred under the strict molecular clock model, whereas substitution rate among sites was calculated with the general time-reversible model (gtr) combined with the gamma parameter and proportion of invariant sites (gtr+Γ+i ) model. mcmc was run for , , steps and sampled every steps and the , first steps of each run were discarded. beast format files were obtained in the provided beauti graphical interface and the generated trees were visualized with the figtree . . . program. finally, statistical analysis was carried out in the tracer package [ ] . ct - , and from the colombian government and the instituto nacional de salud resources bogotá d.c.,colombia. viral vector core and gene therapy current address: robert koch institute at: origins of dengue type viruses associated with increased pathogenicity in the americas dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem dengue viral infections; 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miguel andrés páez for reviewing the manuscript and lissethe pardo for technical assistance at instituo nacional de salud; rive/cyted (red iberoamericana de virosis emergentes) allowed the authors to meet with several other researchers in the field. this research was supported by instituto colombiano para el desarrollo de la ciencia y la tecnología francisco josé de caldas -colciencias grants authors' contributions jamr contributed to the experimental design, carried out the experiments and phylogenetic and molecular clock analysis, and wrote the manuscript. jauc contributed to the experimental design, carried out the experiments and provided a critical review of the manuscript. cdc participated in the experimental design, contributed to the interpretation of data and the critical review of the manuscript. gjrb contributed to the experimental design and provided a critical review of the manuscript. jas contributed with phylogenetic and molecular clock analysis and beast running and provided a critical review of the manuscript. atm conceived the study, its experimental design and provided a critical review of the manuscript. jcgg conceived the study, participated in its design and coordination and provide a final review of the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- -mwj qby authors: mackay, ian m.; arden, katherine e. title: mers coronavirus: diagnostics, epidemiology and transmission date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: mwj qby the first known cases of middle east respiratory syndrome (mers), associated with infection by a novel coronavirus (cov), occurred in in jordan but were reported retrospectively. the case first to be publicly reported was from jeddah, in the kingdom of saudi arabia (ksa). since then, mers-cov sequences have been found in a bat and in many dromedary camels (dc). mers-cov is enzootic in dc across the arabian peninsula and in parts of africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. the ksa is the focal point of mers, with the majority of human cases. in humans, mers is mostly known as a lower respiratory tract (lrt) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in % to % of those infected. however, mers-cov has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. older males most obviously suffer severe disease and mers patients often have comorbidities. compared to severe acute respiratory syndrome (sars), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, mers progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. most human cases of mers have been linked to lapses in infection prevention and control (ipc) in healthcare settings, with approximately % of all virus detections reported among healthcare workers (hcws) and higher exposures in those with occupations that bring them into close contact with camels. sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics have been available almost from the start of the emergence of mers. while the basic virology of mers-cov has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. an email from dr ali mohamed zaki, an egyptian virologist working at the dr soliman fakeeh hospital in jeddah in the kingdom of saudi arabia (ksa) announced the first culture of a new coronavirus to the world. the email was published on the website of the professional emerging diseases (promed) network on th september [ ] (fig. ) and described the first reported case, a year old man from bisha in the ksa. this information led to the rapid discovery of a second case of the virus, this time in an ill patient in the united kingdom, who had been transferred from qatar for care [ ] . the new virus was initially called novel coronavirus (ncov) and subsequentlty entitled the middle east respiratoy syndrome coronavirus (mers-cov). as of nd of september , there have been , detections of viral rna or virus-specific antibodies across countries (additional file : figure s ) confirmed by the world health organization (who), with over a third of the positive people dying (at least , %) [ ] . since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over % of adult dromedary camels (dc; camelus dromedarius) in the ksa [ ] , also dcs across the arabian peninsula and parts of africa that are a source of dc imports for the ksa [ ] . to date, mers-cov has not been detected in dcs tested in zoos or herds from other parts of the world [ ] [ ] [ ] [ ] . occasionally, virus is transmitted from infected dcs to exposed humans. subsequent transmission to other humans requires relatively close and prolonged exposure [ ] . the first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [ , ] . however, sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [ ] . subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase (dpp ; also called cd ); that mers-cov has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than sars-cov; is widespread in dcs; has the potential to infect other animals and that mers kills its human host more often than sars did ( - % versus % for sars [ ] ) [ ] [ ] [ ] [ ] [ ] . in humans, overt disease was given the name middle east respiratory syndrome, with the acronym mers. from intermittent animal-to-human spill-over events, the mers-cov spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. the spread of mers-cov among humans has often been associated with outbreaks in hospitals, with around % of all cases to date involving healthcare workers (hcws). although dcs appear to suffer the equivalent of a 'common cold' from mers-cov infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to % of reported cases. it has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [ ] . studies have established that the mean incubation period for mers is five to six days, ranging from two to days, with to days between when illness begins in one person and subsequently spreads to another [ ] [ ] [ ] [ ] . among those with progressive illness, the median time to death is to days, ranging from five to days [ , ] . fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [ , ] . the first who case definition [ ] defined probable cases of mers based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (lrt) involvement. it also included roles for contact with a probable or confirmed case or for travel or residence within the arabian peninsula. if strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [ ] . from july , the revised who case definition included the importance of seeking out and understanding the role of asymptomatic cases and from june , the who definition more clearly stated that a confirmed case included any person whose sample was rt-pcr positive for mers-cov, or who produced a seroconversion, irrespective of clinical signs and symptoms. [ ] [ ] [ ] apart from the who and the ksa ministry of health reports, asymptomatic or subclinical cases of mers-cov infection were documented in the scientific literature although not always as often as occurred early on [ , ] . the ksa definition of a case became more strict on th may , relying on the presence of both clinical features and laboratory confirmation [ ] . testing of asymptomatic people was recommended against from december [ ] , reinforced by a case definition released by the ksa ministry of health in june [ ] . the ksa has been the source of % of human cases. severe mers is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [ ] [ ] [ ] . interestingly in june , an outbreak in south korea followed a similar distribution [ , ] . among laboratory confirmed cases, fever, cough and upper respiratory tract (urt) signs and symptoms usually occur first, followed within a week by progressive lrt distress and lymphopaenia [ ] . patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [ , ] . disease can progress to acute respiratory distress syndrome and multiorgan system failure [ ] . mers has reportedly killed approximately % of all reported cases, % of cases in the ksa, yet only % of cases in south korea, where mortality ranged from % among younger age groups to % among those aged years and above [ ] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [ ] . general supportive care is key to managing severe cases [ ] . children under the age of years are rarely reported to be positive for mers-cov, comprising only . % (n = ) of total reported cases. between st september and nd december , a study described the then tally of paediatric cases in the ksa, which stood at (two to years of age; median years); nine were asymptomatic ( %) and one infant died [ ] . in amman, jordan, , samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for mers-cov rna, despite being collected at a similar time to the first known outbreak of mers-cov in the neighbouring town of al-zarqa [ ] . a second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not rt-rtpcr positive, the mother did subsequently develop antibodies to mers-cov, suggestive of recent infection [ ] . her exposure history to a mers-cov rt-rtpcr positive relative and an antibody-reactive husband, her incubation period and her symptom history met the who criteria for being a probable mers-cov case [ ] . diagnostic methods were published within days of the promed email announcing the first mers case [ ] , including several now gold standard in-house rt-rtpcr assays (fig. ) as well as virus culture in vero and llc-mk cells [ , , ] . a colorectal adenocarcinoma (caco- ) epithelial cell line has since been recommended for isolation of infections mers-cov [ ] . we previously [ ] .). open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (utr; grey rectangles). fs-frame-shift. predicted regions encompassing recombination break-points are indicated by orange pills. created using geneious v . [ ] and annotated using adobe illustrator. beneath this is a schematic depicting the location of rt-pcr primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest rt-rtpcr screening assays and conventional, semi-nested (three primers) rt-pcr confirmatory sequencing assays [ , ] . publication order is noted by first [ th september ; red] and second [ th december ; orange] coloured rectangles; both from corman et al. [ , ] those assays recommended by the who are highlighted underneath by yellow dots [ ] . the nseq reverse primer has consistently contained one sequence mismatch with some mers-cov variants. an altered version of that from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res vol : - with permission from elsevier [ ] reviewed the broad tropism of mers-cov [ ] . however, as is well described, cell culture is a slow, specialised and insensitive method [ ] while pcr-based techniques are the preferred method for mers-cov detection. the first open reading frames (orf a and b; fig. ) have become a key diagnostic and taxonomic target for cov species identification. with less than % identity between the amino acid sequence of mers orf ab and betacoronavirus relatives, tylonycteris bat hku and pipistrellus bat hku , it can be concluded that it is a novel and distinct virus. mers-cov is predicted to encode ten open reading frames with ' and ' untranslated regions [ ] . the structural proteins include the spike (s), envelope (e), membrane (m) and nucleocapsid (n) [ ] . the products of orf a and orf b are predicted to encode nonstructural proteins. the majority of specimen testing to date has employed validated rt-rtpcr assays shown to be sensitive and specific [ , , ] . the realstar® kit uses these whorecommended assays [ ] . the target sequences of these screening assays have not changed among genomes examined until at least mid- (imm observation). other rt-rtpcr assays have been developed and validated for use as laboratory-based diagnostic tools [ ] [ ] [ ] . additionally, loop-mediated [ , ] or recombinase polymerase [ ] isothermal assays have been designed for field deployment. the detection of mers-cov antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. detection of viral proteins rather than viral rna indicates the likely presence of infectious virus. the first rapid immunochromatographic tool described could detect recombinant mers-cov nucleocapsid protein from dc nasal swabs with % sensitivity and % specificity compared to rt-rtpcr [ ] . a different approach used a monoclonal antibody-based capture elisa targeting the mers-cov nucleocapsid protein with a sensitivity of tcid and % specificity [ ] . demonstration of a seroconversion to a mers-cov infection meets the current who definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior mers-cov infection and help support transmission studies. because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral rna [ ] . strategic, widespread sero-surveys of humans using samples collected after are infrequent. much of the arabian peninsula and all of the horn of africa lack baseline data describing the proportion of the community who may have been infected by a mers-cov. however, sero-surveys have had widespread use in elucidating the role of dcs as a transmission source for mers-cov. because of the identity shared between dc and human mers-cov (see molecular epidemiology: using genomes to understand outbreaks), serological assays for dc sero-surveys should be transferrable to human screening with minimal re-configuration. also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested mers-cov isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single mers-cov serotype [ ] . the development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to mers-cov, as well as to likely sources of cross-reaction [ ] . obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [ ] . a number of commercial elisa kits, immunofluorescent assays (ifa) kits, recombinant proteins and monoclonal antibodies have been released [ , [ ] [ ] [ ] [ ] . initially, conventional ifas were used for human sero-surveys. these relied on mers-cov-infected cell culture as an antigen source, detecting the presence of human anti-mers-cov igg, igm or neutralizing antibodies in human samples [ , , ] . no sign of mers-cov antibodies was found among , sera from patients visiting hospital in jeddah, from through , prior to the description of mers-cov [ ] . nor did ifa methods detect any sign of prior mers-cov infection among a small sample of healthy blood donors from another hospital in jeddah (collected between jan and dec ) [ ] . of slaughterhouse workers, only eight ( . %) were positive by ifa, and those sera could not be confirmed by virus neutralization (nt) test. the study indicated that hcov-hku was a likely source of crossreactive antigen in the whole virus ifa [ ] . whole virus mers-cov ifa also suffered from some cross-reactivity with convalescent sars patient sera and this could not be resolved by an nt test which was also cross-reactive [ ] . ifa using recombinant proteins instead of whole-virus ifa, has been shown to be a more specific tool [ ] . since asymptomatic zoonoses have been posited [ ] , an absence of antibodies to mers-cov among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering dcs [ ] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. ifa using recombinant proteins instead. some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the mers-cov nucleocapsid and spike proteins [ , ] , or using a recombinant lentivirus expressing mers-cov spike protein and luciferase [ , ] . a pseudo particle neutralization (ppnt) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (mnt) test. [ , , [ ] [ ] [ ] ] studies using small sample numbers and ppnt found no evidence of mers-cov neutralizing antibody in sera from children with lrt infections between may and may , sera from to year old male blood donors and selfidentified animal workers from the jazan region of the ksa during [ , ] . similarly, a study of four herdsmen in contact with an infected dc herd in al-ahsa, eight people who had intermittent contact with the herd, veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in al-ahsa and controls who were not exposed to dcs in any professional role, found none with serological evidence of past mers-cov infection using the ppnt assay [ ] . a delay in the neutralizing antibody response to mers-cov infection was associated with increased disease severity in south korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [ ] . the implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. a jordanian outbreak of acute lrt disease in a hospital in was retrospectively found to be associated with mers-cov infection, initially using rt-rtpcr, but subsequently, and on a larger scale, through positivity by elisa and ifa or mnt test. [ , , ] this outbreak predated the first case of mers in the ksa. the elisa used a recombinant nucleocapsid protein from the group betacoronavirus bat-cov hku to identify antibodies against the equivalent crossreactive mers-cov protein [ ] . it was validated using sera collected from people with prior hcov-oc , hcov- e, sars-cov, hcov-nl , hrv, hmpv or influenza a(h n ) infections but was reportedly less specific than the recombinant ifa discussed above. it was still considered an applicable tool for screening large sample numbers [ ] . a protein microarray expressing the s protein subunit has also been validated and widely used for dc testing [ , ] . detection of mers-cov infection using elisa or s subunit protein microarray [ ] is usually followed by confirmatory ifa and/ or a plaque-reduction neutralization (prnt) [ , , ] or mnt test. [ , , ] this confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in dcs (bovine cov, bcov) or humans (hcov-oc , hcov- e, hcov-nl , hcov-hku , sars-cov). in the largest study of human sera, a tiered diagnostic process assigned both recombinant ifa and recombinant elisa positive sera to 'stage ' seropositivity. a stage seropositive result additionally required a suitably titred prnt result [ ] . the study found sera collected in to from , ( . %) people in ksa provinces contained mers-cov antibodies, but significantly higher proportions in occurred in camel shepherds (two of ; . %) and slaughterhouse workers (five of ; . %) [ ] . contemporary surveys are needed. mers-cov does not appear to be easily transmitted from dcs to humans, or perhaps it is [ ] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. this was reinforced when a false positive us case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, nt assay and was subsequently retracted [ , ] . the who recommends sampling from the lrt for mers-cov rt-rtpcr testing, especially when sample collection is delayed by a week or more after onset of symptoms. [ ] lrt samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [ ] . recommended sample types include bronchoalveolar lavage (bal), tracheal/tracheobronchial aspirate, pleural fluid and sputum [ , ] . fresh samples yield better diagnostic results than refrigerated material [ ] and if delays in testing of ≥ h are likely, samples (except for blood) should be frozen at − °c [ ] . if available, lung biopsy or autopsy tissues can also be tested [ ] . the urt is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when urt sampling is to be conducted [ ] . paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first - days if conducting rt-rtpcr [ , ] . human urine and stool have been found to contain mers-cov rna to days after symptom onset [ , , ] and are listed as samples that should be considered [ , ] . in two cases that arrived in the netherlands, urine was rt-rtpcr negative but faeces was weakly positive and sera were rt-rtpcr positive for five days or more [ ] . the finding of mers-cov viral rna in serum provides an avenue for retrospective pcr-based studies if respiratory samples are unavailable [ ] . rnaaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [ ] . clinically suspected mers cases may return negative results by rt-rtpcr. data have shown one or more negative urt samples may be contradicted by further urt sampling or the use of lrt samples, which is preferred [ , , ] . higher viral loads occur in the lrt compared to the urt. [ , , , ] this fits with the observation that the majority of disease symptoms are reported to manifest as systemic and lrt disease [ ] . however, on occasion, even lrt specimens from mers cases may initially be negative, only to later become positive by rt-pcr [ ] . this may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [ ] . despite this both the largest human mers-cov studies [ , [ ] [ ] [ ] and smaller ones [ , , ] , use samples from the urt. it is then noteworthy that one study reported an association between higher loads in the urt and worse clinical outcome including intensive care and death [ ] . at writing, no human data exist to define whether the virus replicates solely or preferentially in the lrt or urt, or replicates in other human tissues in vivo although mers-cov rna has been detected from both the urt and lrt in a macaque monkey model [ ] .the distribution of dpp in the human upper airways is also not well described. individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [ , , , ] in one instance, a hcw shed viral rna for days in the absence of disease [ ] . it is an area of high priority to better understand whether such cases are able to infect others. over three quarters of mers cases shed viral rna in their lrt specimens (tracheal aspirates and sputum) for at least days, while only % of contacts were still shedding rna in their urt specimens [ , ] . in the only study to examine the effect of sample type on molecular analysis, nasopharyngeal aspirates (npa; an urt sample), tracheal aspirates, sputa and three bal were examined. the tracheal aspirates and bal returned the highest viral load values followed by npa and sputum. unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in npa testing, were significantly correlated with severe disease and death [ , , ] . this study demonstrated the importance of lrt sampling for whole genome sequencing. when tested, samples positive for mers-cov are often negative for other pathogens [ , , , ] . however, many studies make no mention of additional testing for endemic human respiratory viruses [ , , , ] . when viruses are sought, they have included human herpesvirus (hhv), rhinoviruses (hrv), enteroviruses (ev), respiratory syncytial virus (rsv), parainfluenzavirus types , and (pivs),influenzaviruses (ifvs), endemic hcovs, adenoviruses (advs) metapneumovirus (mpv) and influenza a\h n virus; co-detections with mers-cov have been found on occasion [ , , , , ] . bacterial testing is sometimes included (for example, for legionella and pneumococcus) but the impact of bacterial co-presence is also unclear [ , [ ] [ ] [ ] . further testing of the lrt sample from the first mers case used ifa to screen for some viruses (negative for ifv, pivs, rsv and advs) and rt-pcr for others (negative for adv, evs, mpv and hhvs) [ ] . rt-pcr also detected mers-cov. the who strongly recommends testing for other respiratory pathogens [ ] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both mers cases and their contacts. little is known of other causes of mers-like pneumonia in the ksa or of the general burden of disease due to the known classical respiratory viruses. testing of adult pilgrims performing the hajj in to has not detected any mers-cov. in , nasal swabs from pilgrims collected prior to leaving for or departing from the ksa were tested [ ] . in , testing was significantly scaled up with , nasopharyngeal swabs from , incoming pilgrims and , swabs from outgoing pilgrims tested [ ] . it should be noted that most pilgrims arrived from mers-free countries. a further swabs were taken from pilgrims with influenza-like illness [ , ] . in earlier hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [ ] [ ] [ ] over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among hajj pilgrims. [ ] a lrt sample is often not collected for these studies [ , , ] , so false negative findings are a possibility although little is known about the initial site of mers-cov infection and replication; it may have been assumed it was the lrt because disease was first noticed there but the urt may be the site of the earliest replication. in jeddah between march and july (hereafter called the jeddah- outbreak; fig. ), there was a rapid increase in mers cases, accompanied by intense screening; approximately , samples from in and around the region were tested in a month yielding around mers-cov detections (~ % prevalence) [ ] . among , individuals sampled and tested across the ksa between october and september , ( . %) detections were made in a hospital-centric population which included hospitalized cases (n = , ; . %), their families (n = ; . %) and associated hcws (n = , ; . %) [ ] . among the detections, ( . %) were hcws and ( . %) were family contacts [ ] . the - % prevalence of active mers-cov infections is not dissimilar to the hospital-based prevalence of other human covs. [ ] however, the proportion of deaths among those infected with mers-cov is much higher than that known for the hcovs nl , hku , e or oc in other countries, and even above that for sars-cov; it is not a virus that could reasonably be described as a "storm in a teacup". it is the low transmission rate that has prevented worldwide spread, despite many "opportunities". very early in the mers outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of mers-cov with three of the first five cases having contact with dcs [ , , ] . today, animal mers-cov infections must be reported to the world organization for animal health as an emerging disease [ ] . a summary of the first mers cases reported by the who defined animal contact with humans as being direct and within days prior to symptom onset [ ] . this definition made no specific allowance for acquisition from dcs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [ ] . camels are known to produce high levels of mers-cov rna in their urt and lungs [ ] . providing support for a droplet transmission route and perhaps indicating the presence of rna in smaller, drier droplet nuclei, mers-cov rna was identified in a high volume air sample collected from a barn housing an infected dc [ ] . the precise source from which humans acquire mers-cov remains poorly studied but it seems likely that animal and human behavioural factors may play roles (fig. ) [ ] . these factors may prove important for human cases who do not describe any dc contact [ ] nor any contact with a confirmed case. whether the who definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. wording focuses on consumption of dc products but does not specifically ascribe risk to a droplet route for acquisition of mers-cov from dc [ ] . some mers patients are listed in who disease notices as being in proximity to dcs or farms, but the individuals have not described coming into contact with the animals. no alternative path for acquiring infection is reported in many of these instances. what constitutes a definition of "contact" during these interviews has been defined for one study [ ] . despite this lack of clarity, the who consider that evidence linking mers-cov transmission between dcs to humans is irrefutable (fig. ) [ ] . the possibility that bats were an animal host of mers-cov was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [ ] [ ] [ ] [ ] . conclusive evidence supporting bats as a source for human infections by mers-cov has yet to be found, but bats do appear to host ancestral representatives [ , ] . however, these are not variants of the same virus nor always within the same phylogenetic lineage as mers-cov; they are each a genetically distinct virus. bat-to-human infection by mers-cov is a purely speculative event. the only piece of mers-cov-specific evidence pointing to bats originates from amplification of a nt fragment of the rnadependent rna polymerase gene of the mers-cov genome, identified in a faecal pellet from an insectivorous emballonuridae bat, taphozous perforatus found in bisha, the ksa [ ] . while very short, the sequence of the fragment defined it as a diagnostic discovery. subsequently a link to dcs was reported [ ] and that link has matured into a verified association [ , ] (fig. ) . (see figure on previous page.) fig. monthly detections of mers-cov (blue bars) and of cases who died (red bars) with some dates of interest marked for to th september . an approximation of when dc calving season [ ] and when recently born dcs are weaned is indicated. spring (green) and summer (orange) in the arabian peninsula are also shaded. note the left-hand y-axis scale for and which is greater than for / . sources of these public data include the who, ministries of health and flutrackers [ ] [ ] [ ] . earlier and subsequent versions of this chart are maintained on a personal blog [ ] . modified and reprinted from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res vol : - with permission from elsevier [ ] dcs, which make up % of all camels, have a central presence in the arabian peninsula where human-dc contact ranges from little to close [ ] . contact may be commonplace and could occur in variety of ways (fig. a) . there are several large well-attended festivals, races, sales and parades which feature dcs and dcs are also kept and bred close to populated areas in the ksa [ , ] . dc milk and meat are widely consumed and the older dc is an animal of ritual significance after the hajj pilgrimage [ ] . however, mers-cov infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing dc products. daily ingestion of fresh unpasteurized dc milk is common among the desert bedouin and many others in the ksa. dc urine is also consumed or used for supposed health benefits. despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among mers cases; this may simply be a reporting issue rather than an unexplainable absence of mers. a small case-control study published in identified direct dc contact, and not ingestion of products, to be associated with onset of mers [ ] . the first sero-survey of livestock living in the middle east region was conducted during - [ ] . dcs were sampled from a mostly canary island-born herd and from omani dcs (originally imported from the horn of africa) [ ] . a neutralising antibody assay found only % of strongly seropositive canary island [ ] . b camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor ipc in healthcare settings where transmission is amplified, accounting for the bulk of cases. there are human mers cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) mers-cov infection may be implicated in transmission dc sera could neutralise mers-cov while all omani dc sera had high levels of specific mers-cov neutralizing antibody [ ] . this indicated that dcs had in the past been infected by mers-cov, or a very similar virus. since this study, a host of peer-reviewed reports have looked at both dcs and other animals, and the possibility that they may host mers-cov infection. seropositive dcs have been found throughout the arabian peninsula including oman, the ksa, qatar, jordan, the united arab emirates (uae), kuwait as well as sudan, somalia, egypt, tunisia, nigeria, kenya and ethiopia in africa and the canary islands [ , [ ] [ ] [ ] [ ] [ ] . other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, bactrian camels, llamas and guanaco (south american camelids) but none had detectable neutralising antibody against mers-cov [ , , , , , , ] . no virology or serology studies of human samples from areas in africa where there are camels with a history of mers-cov have been reported to date. however,an absence of unexplained pneumonia that may be attributable to mers-cov infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. it is thus unclear whether mers-cov, or an antigenically related cov, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the arabian peninsula [ ] . mers-cov rna has also been detected in dc samples, and recovery of infectious virus has also been achieved from dc samples [ , , , , [ ] [ ] [ ] [ ] [ ] . from some of these, full or majority length genomes of mers-cov have been sequenced [ , , ] . dc versions of mers-cov were found to be as similar to each other, as were variants detected from different humans over time and across distance. antibody screening assays have also detected crossreactive antibodies in sera. these were identified as such by screening sera against similar viruses, for example bcov or hcov-oc (as an antigenic facsimile for bcov). it is possible that other mers-cov-like viruses also reside within dcs, but this does not detract from the definitive finding of mers-cov genetic sequences in both dcs and humans [ , , ] . screening studies have shown that juvenile dcs are more often positive for virus or viral rna while older dcs are more likely to be seropositive and rna or virus negative [ , , ] . in adult dcs, mers-cov rna has been detected among animals with pre-existing antibody, suggesting re-infection is possible [ , ] . viral loads among positive dcs can be very high [ , , , , ] and dcs have been found positive both when ill with urt respiratory signs [ , , , ] or when apparently healthy [ ] . these findings indicate dcs host natural mers-cov infections. furthermore, stored dc sera have revealed signs of mers-cov in dcs which date back over three decades (the earliest collected in ) [ , , ] . older sera have not been tested and so precisely how long dcs have been afflicted by mers-cov, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in africa or the arabian peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. researchers sought to determine a direction for infection; were dcs transmitting virus to humans or were humans infecting dcs? at a qatari site, a farm owner and his employee became ill in mid-october and tested positive for mers-cov rna in a sputum and throat swab sample, respectively. rt-rtpcrs found mers-cov rna in of positive dc nasal swabs at the farm; six ( %) positive by two or more assays [ ] . the results indicated a recent outbreak had occurred in this herd; the first indication of mers-cov rna found within dcs with a temporal association to human infections. three positive dc samples were confirmed by sequencing a nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and dc mers-cov sequences [ ] . the dcs and human contacts yielded orf a and orf b sequences differing by only a single nucleotide each, clustering closely with the hafr-al-batin_ _ variant [ ] . subsequent case studies found evidence of a concurrent human and dc infection and the direction of that infection was inferred to be from the ill dcs and to their human owners [ , , ] . partial genome sequences indicated that a human and a mers-cov rt-rtpcr positive dc had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [ ] all nine dc in the owner's herd, serially sampled, reacted in a recombinant s antigen elisa, with the two animals that had been rt-rtpcr positive showing a small, verifiable rise in antibody titre [ ] . a rise in titre theoretically begins to days after dc infection [ ] . the authors suggested that the rise in titre in dc sera which occurred alongside a declining rna load, while the patient was actively ill and hospitalized, indicated that the dcs were infected first followed by the owner [ , ] . bcov antibodies were also present, and rising in one of the two rt-rtpcr positive animals but no animal's antibodies could neutralise bcov infection [ ] . camel calving season occurs in the winter months (between late october and late february; fig. ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve dc populations [ ] . what role maternal camel antibody might play in delaying infection of calves remains unknown [ , ] . juvenile dcs appear to host active infection more often than adult dcs and thus the sacrificial slaughter of dcs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. in contrast to earlier results, slaughterhouse workers who kill both younger and older dcs, may be an occupational group with significantly higher incidence of seropositivity to mers-cov when animals have active mers-cov infections [ , , [ ] [ ] [ ] . expanded virological investigations of african dcs may lead to more seropositive animals and geographic areas in which humans may be at risk. it is possible that there are areas where humans already harbour mers-cov infections that have not been identified because of an absence of laboratory surveillance. virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [ , ] . infectious mers-cov added to dc, goat or cow milk and stored at °c could be recovered at least h later and, if stored at °c, recovery was possible for up to h [ ] . mers-cov titre decreased somewhat when recovered from milk at °c but pasteurization completely ablated mers-cov infectivity [ ] . in a subsequent study, mers-cov rna was identified in the milk, nasal secretion and faeces of dcs from qatar [ ] . a single study has examined the ability of mers-cov to survive in the environment [ ] . plastic or steel surfaces were inoculated with tcid of mers-cov at different temperature and relative humidity (rh) and virus recovery was attempted in cell culture. at high ambient temperature ( °c) and low rh ( %) mers-cov remained viable for h [ ] . by comparison, a well known and efficently transmitted respiratory virus, influenza a virus, could not be recovered in culture beyond four hours under any conditions [ ] . aerosol experiments found mers-cov viability only decreased % at low rh at °c. in comparison, influenza a virus decreased by % [ ] . mers-cov survival is inferior to that previously demonstrated for sars-cov [ ] . for context, pathogenic bacteria can remain viable and airborne for min in a coughed aerosol and can spread m. mers-cov's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [ ] . whether mers-cov can remain adrift and infectious for extended periods (truly airborne) remains unknown. such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. the nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the al-ahsa hospital, the ksa and the south korean outbreaks [ , , , ] . by extrapolation, aerosol-generating events involving dcs (urination, defecation, and preparation and consumption of dc products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. the provision of evidence supporting the best formulation of personal protective equipment to be worn by hcws who receive, manage or conduct procedures on infectious cases remains a priority. mers-cov was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to mers-cov transmission chains. however, transmission of mers-cov is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [ , , , , , , ] in a household study, of ( %) contacts of mers-cov positive index patients were rna or antibody positive; the rate of general transmission, even in outbreaks is around % [ ] . it seems that the majority of human cases of mers-cov, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of mers-cov has not been self-sustaining [ ] [ ] [ ] [ ] [ ] . that is to say, the basic reproduction number (r ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. if r was greater than , a sustained increase in case numbers would be expected. some r o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. that mers has had a constant presence in the arabian peninsula since is due to ongoing, sporadic spill-over events from dcs amplified by poorly controlled hospital outbreaks. the first known mers human-to-human transmission event was one characterized by acute lrt disease in a healthcare setting in jordan. in stark contrast, a sero-survey of hcw who were sometimes in close and prolonged contact with the first, fatal mers-cov case in [ ] , found none of the hcw had seroconverted four months later, despite an absence of eye protection and variable compliance with required ppe standards [ ] . early on in the mers story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. contacts of confirmed mers cases were often observed for clinical illness, but not tested. these omissions may have confounded our understanding of mers-cov transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. case-control studies were not a focus. as testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [ ] . a rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [ ] ) resulted in a drop in the proportion of fatal cases during the jeddah- outbreak. historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. upon follow-up, over three-quarters of such mers-cov rna positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [ ] raising some question over the reliability of other reported data. the proportion of fatal mers cases within the ksa compared to outside the ksa, as well as the age, and sex distribution change in different ways when comparing mers outbreaks. approximately % of mers cases ( of ) in the ksa were fatal betwen and december while % ( of ) died among those occurring outside of the ksa. the total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. however the proportion of male deaths from total males with mers is a similar figure to that for females. in the ksa, there is a greater proportion of younger males among cases and deaths than were observed from the south korean or the jeddah- outbreaks (additional file : figure s ). why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [ ] . as a group, hcws comprised % of mers cases in the ksa and south korea. it is apparent that the weekly proportion of infected hcws increases alongside each steep rise in overall detections (fig. ) . in may , the who published guidelines for ipc during care of probable or confirmed cases of mers-cov infection in a healthcare setting [ ] . this is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [ ] . these rises in mers-cov detections can decrease the average age during each event because hcws are usually younger than inpatients with mers. healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (ipc) procedures [ , ] . most of the analysis of mers-cov genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [ ] [ ] [ ] . mers-cov was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. the technique can produce genomic [ ] [ ] [ ] . earlier and subsequent versions of this chart are maintained on a personal blog [ ] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [ , ] . despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during mers-cov characterization [ ] . as more genomes from both humans and dcs have been characterized, two clades have become apparent; a and b (fig. ) . clade a contains only human-derived mers-cov genomes from jordan, while clade b comprises the majority of human and camel genomes deduced thus far [ ] . two studies during , one looking at jeddah- mers-cov variants and another looking at a variant exported from south korea to china, have now identified signs of genetic recombination among mers-cov variants. while human and camel whole genome sequences have retained > % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [ ] [ ] [ ] . shared identity implies that the major source for human acquisition is the dc, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. over a month, a dc virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that dcs are the natural, rather than intermediate, host for the mers-cov we know today [ ] . to date, recombination has been localised to breakpoints near the boundary between orf a and orf b regions, within the spike gene [ ] and in the orf b region (fig. ) [ ] . it is not unexpected that recombination should occur since it is well known among other covs [ ] and because the majority of mers-cov whole genomes collected from samples spanning three years ( - ) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [ , , , , , [ ] [ ] [ ] . changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. if we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely fig. the genetic relationship between mers-cov nucleotide sequences (downloaded from genbank using the listed accession numbers and from virological.org [ ] ). this neighbour joining tree was created in mega v using an alignment of human and dcderived mers-cov sequences (geneious v . [ ] ). clades are indicated next to dark (clade a) or pale (clade b) blue vertical bars. camel icons denote genomes from dcs. healthcare or community outbreaks are boxed and labelled using previously described schemes [ , ] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. genetic mutations noted during the largest of human outbreaks, jeddah- , did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [ , ] . however, we understand very little of the phenotypic outcomes that result from subtle genetic change in mers-cov genomes. to date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [ ] . but vigilance and larger, more contemporary and in vivo studies are needed. genome sequence located to a distinct clade were identified from an egyptian dc that was probably imported from sudan. this does not fit into either of the current clades [ , , ] . a virus sequenced from a neoromicia capensis bat was more closely related to mers-cov than other large bat-derived sequences had been to that point, but the genome of a variant of a mers-cov has yet to be discovered and deduced from any bat [ ] . analyses of mers-cov genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (fig. ) , which encodes the spike protein and accessory proteins [ ] . at least nine mers-cov genomes contained amino acid substitutions in the receptor binding domain (rbd) of the spike protein and codons (n-terminal region), (rbd), (in heptad repeat ), and bear investigation as markers of adaptive change [ , ] . the spike protein had not changed in the recombinant mers-cov genome identified in china in but was reported to have varied at a higher rate than that for complete mers-cov genomes, among south korean variants [ , ] . this highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. despite this, one assay amplifying a nucleotide fragment of the spike s domain gene for sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [ ] . genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (fig. ) [ ] . this approach was employed when defining the geographically constrained mers hospital outbreak in al-ahsa, which occurred between st april and rd may , as well as clusters in buraidah and a community outbreak in hafr al-batin, the ksa. genomic sequencing identified that approximately mers-cov detections from a community outbreak in hafr al-batin between june and august may have been triggered by an index case becoming infected through dc contact [ ] . sequencing mers-cov genomes from the al-ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [ ] . however, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [ ] . in riyadh- , genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [ , , ] . riyadh is a nexus for camel and human travel and has had more mers cases than any other region of the ksa to date but also harbours a wide range of mers-cov variants [ , , ] . however the south korean outbreak originated from a single infected person, resulting in three to four generations of cases [ , ] . studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [ ] . for many mers cases detected outside the arabian peninsula, extensive contact tracing has been performed and the results described in detail. contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. results of contact tracing to date have found that onward transmission among humans is an infrequent event. for example, there were contacts, both symptomatic and asymptomatic, of a case treated in germany who travelled from the uae but no sign of virus or antibody were found in any of them [ ] . the very first mers case had made contact with hcws and others, but none developed any indication of infection [ ] . in a study of contacts of a case treated in france, only seven matched the definition for a possible case and were tested; one who had shared a m hospital room while in a bed . m away from the index case for a prolonged period was positive [ ] . none of the contacts of the first two mers cases imported into the usa in contained any mers-cov footprint [ ] and none of the contacts of two travellers returning to the netherlands developed mers-cov antibodies or tested rna positive [ , ] . analyses of public data reveal many likely instances of nosocomial acquisition of infection in the arabian peninsula and these data may be accompanied by some details noting contact with a known case or facility. one example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [ ] . contact tracing was a significant factor in the termination of a outbreak involving multiple south korean hospitals [ ] . such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (fig. c) . the hospital-associated outbreak in jeddah in was the largest and most rapid accumulation of mers-cov detections to date. the greatest number of mers-cov detections of any month on record occurred in jeddah in april. the outbreak was mostly (> % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [ , , ] . a rise in fatalities followed the rapid increase in case numbers. in two large outbreaks occurred. south korea was the site of the first large scale outbreak outside the arabian peninsula and produced the first cases in both south korea and china, occurring between may and july . this was closely followed by a distinct outbreak in ar riyad province in the ksa which appeared to come under control in early november. after staying in bahrain for two weeks, a year old male ( m) travelled home to south korea via qatar, arriving free of symptoms on the th may [ ] . he developed fever, myalgia and a cough nearly a week later ( th ). he visited a clinic as an outpatient between the th and th of may and was admitted to hospital a on the th [ ] . he was discharged from hospital a on the th then visited and was admitted to the emergency department of hospital b on the th . during this second stay, a sputum sample was taken and tested positive for mers-cov on the th [ , ] , triggering transfer to the designated isolation treatment facility. over a period of days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the south korean outbreak. approximately people were infected during this time [ ] . in total cases were generated in this outbreak, all linked through a single transmission chain to m; cases died [ ] . in south korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [ ] . other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with mers, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of korean society, crowded emergency rooms, poor ipc measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [ , [ ] [ ] [ ] . all of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [ , , , [ ] [ ] [ ] . few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. the hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [ , [ ] [ ] [ ] . early in the outbreak, a infected traveller, the son of an identified case in south korea, passed through hong kong on his way to china where he was located, isolated and cared for in china [ , , ] . no contacts became ill. the outbreak was brought under control in late july/ early august [ ] after improved ipc measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [ , ] . a review of public data showed that, as for mers in the ksa, older age and the presence of underlying disease were significantly associated with a fatal outcome in south korea. [ ] even though r is < , super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over , such as this one, are not unexpected from mers-cov infection [ ] . the dynamic of an outbreak depends on the r and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [ ] . in the region of ar riyad, including the capital city of riyadh, a hospital based cluster began, within a single hospital, from late june [ ] . by mid-september there had been approximately cases reported but the outbreak appeared to been brought under control in november. it became apparent early on that mers-cov spread relatively ineffectively from human-to-human. despite ongoing and possibly seasonal introduction of virus to the human population via infected dcs and perhaps other animals yet to be identified, the vast majority of mers-cov transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. this opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. it remains unclear if this group are uniquely affected by mers-cov or if other respiratory virus infections, including those from hcovs, produce a similarly serious impact. in south korea, a single imported case created an outbreak of cases and deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. household human-to human transmission occurs but is also limited. educational programs will be essential tools for combatting the spread of mers-cov both within urban and regional communities and for the health care setting. vigilance remains important for containment since mers-cov is a virus with a genetic makeup that has been observed for only three years and is not stable. among all humans reported to be infected, nearly % have died. continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. whole genome sequencing has been used extensively to study mers-cov travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. mers and sars have some clinical similarities but they also diverge significantly [ ] . defining characteristics include the higher pfc among mers cases (above % in and currently at - %; well above the % of sars) and the higher association between fatal mers and older males with underlying comorbidities. for the viruses, mers-cov has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to sars-cov. there appears to be a - % prevalence of mers-cov in the ksa with a % chance of secondary transmission within the household. there is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any r would predict on face value. nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of mers or mers-cov during or immediately after these events. there is no evidence that mers-cov is a virus of pandemic concern. nonetheless, hospital settings continue to describe mers cases and outbreaks in the arabian peninsula. as long as we facilitate the spread of mers-cov among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. the mers-cov appears to be an enzootic virus infecting the dc urt with evidence of recent genetic recombination. it may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in . rt-pcr testing of lrt samples remains the gold standard for mers-cov confirmation. serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. similarly, the important question of whether those who do shed mers-cov rna for extended periods are infectious while appearing well, continues to go unanswered. it is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. while the basic virology of mers-cov has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. additional file : figure s . the severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd absence of mers-coronavirus in bactrian camels, southern mongolia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia ( ) and australia ( ) and characterisation of assay specificity middle east respiratory syndrome coronavirus infection not found in camels in japan absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread lack of middle east respiratory syndrome coronavirus transmission from infected camels tensions linger over discovery of coronavirus as outbreak continues middle east respiratory syndrome coronavirus (mers-cov): evidence and speculations cumulative number of reported probable cases of sars in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection replicative capacity of mers coronavirus in livestock cell lines human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-tohuman transmission case definition for case finding severe respiratory disease associated with novel coronavirus middle east respiratory syndrome coronavirus case definition for reporting to who. interim case definition as of middle east respiratory syndrome coronavirus | case definition for reporting to who revised interim case definition for reporting to who -middle east respiratory syndrome coronavirus (mers-cov): interim case definition as of transmission of mers-coronavirus in household contacts screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study case definition and surveillance guidance for mers-cov testing in saudi arabia infection prevention/control and management guidelines for patients with middle east respitaory syndrome coronavius (mers-cov) infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection middle east respiratory syndrome coronavirus in children characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia preliminary epidemiological assessment of mers-cov outbreak in south korea mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea invited editorial: mers-cov an emerging viral zoonotic disease: three years after and counting middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in stillbirth during infection with middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia development and evaluation of a 'real-time' rt-pcr for the detection of enterovirus and parechovirus rna in csf and throat swab samples mers: emergence of a novel human coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans laboratory testing for middle east respiratory syndrome coronavirus | interim guidance performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus laboratory testing for middle east respiratory syndrome coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses realtime sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in serological assays for emerging coronaviruses: challenges and pitfalls middle east respiratory syndrome coronavirus immunoassays test characteristics: anti-mers-cov elisa camel (igg) recombivirus anti-middle east respiratory syndrome coronavirus (mers-cov) elisa kits ncov / novel coronavirus) nucleocapsid antibody, mouse mab is mers another sars? investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc ( ) by both immunofluorescent and neutralizing antibody tests asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates contact investigation for imported case of middle east respiratory syndrome seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during . influenza other respir viruses kinetics of serologic responses to mers coronavirus infection in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological findings from a retrospective investigation specific serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study cdc concludes indiana mers patient did not spread virus to illinois business associate middle east respiratory syndrome: what clinicians need to know interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov characteristics of traveler with middle east respiratory syndrome distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms health protection agency ukncit. evidence of person-to-person transmission within a family cluster of novel coronavirus infections prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the hajj a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications respiratory tract samples, viral load and genome fraction yield in patients with middle east respiratory syndrome ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response a case of imported middle east respiratory syndrome coronavirus infection and public health response viral respiratory infections among hajj pilgrims in influenza a and b viruses but not mers-cov in hajj pilgrims acute respiratory infections in travelers returning from mers-cov-affected areas changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a -year retrospective analysis of data soaring mers cases cause pandemic jitters, but causes are unclear co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in recovery from severe novel coronavirus infection human isolate . who statement on the fifth meeting of the ihr emergency committee concerning mers-cov | who statement mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus taking stock of the first mers coronavirus cases globally-is the epidemic changing? human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection. zoonoses public health summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in bats, saudi arabia emerging infectious diseases associated with bat viruses bats and their virome: an important source of emerging viruses capable of infecting humans coronavirus diversity, phylogeny and interspecies jumping rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease link between mers virus and camels worries breeders dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) fiqh us-sunnah antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries mers coronavirus neutralizing antibodies in camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai serologic assessment of possibility for mers-cov infection in equids mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus the holy qur'an sacrificing an animal at mina occupational exposure to dromedaries and risk for mers-cov infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels the effects of temperature and relative humidity on the viability of the sars coronavirus viability of pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? an observational, laboratory-based study of outbreaks of mers-coronavirus in jeddah and riyadh, kingdom of saudi arabia assessment of the mers-cov epidemic situation in the middle east region assessing the pandemic potential of mers-cov interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility transmission dynamics and control of ebola virus disease (evd): a review health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures age-specific and sex-specific morbidity and mortality from avian influenza a(h n ) mers-cov outbreak in jeddah-a link to health care facilities infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation middle east respiratory syndrome coronavirus recombination and the evolution of science and public health in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of reliable typing of mers-cov variants with a small genome fragment variations in spike glycoprotein gene of mers-cov molecular epidemiology of hospital outbreak of middle east. respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities followup of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands press release -who, korea-who joint mission on mers cov call for infection control to stem mers infection control and mers-cov in health workers an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome coronavirus (mers-cov) -republic of korea samsung hospital to invest w b in post-mers improvements why the panic? south korea's mers response questioned intensified public health measures help control mers-cov outbreak in the republic of korea south korean mers outbreak spotlights lack of research complete genome sequence of middle east respiratory syndrome coronavirus isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china urgent call for research on middle east respiratory syndrome (mers) in korea middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak no respite for korean economy even as mers patients recover middle east respiratory syndrome coronavirus (mers-cov) -china mers in south korea and china: a potential outbreak threat? objective determination of end of mers outbreak, south korea updates on mers outbreak (as of : on surveillance operation for the st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission moh holds the first press conference to update community and media on mers severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) - case list of moh/who novel coronavirus mers ncov announced cases geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data recent evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china any unreferenced opinions expressed herein are those of the authors and do not necessarily represent the views of any employer or institution. our thanks go to andrew rambaut, maia majumder, marion koopmans and hale abdali for helpful discussions on social media and cmam and riam for patience. the authors declare that they have no competing interests.author's contributions imm and kea contributed equally to this work and read an approved the final manuscript. key: cord- -sdg zdc authors: lin, huixing; chen, lei; gao, lu; yuan, xiaomin; ma, zhe; fan, hongjie title: epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: sdg zdc background: porcine epidemic diarrhea virus (pedv) is the main causative agent of porcine epidemic diarrhea (ped). since december , a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants. methods: a pedv strain, yc , was isolated from intestinal samples of suckling piglets with acute diarrhea in . the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . software. the immune protective efficiency of yc were determined by testing pedv neutralizing antibodies in sera, the colostrum and the milk on th day after farrowing of the immunized sows. the diarrhea symptoms of piglets after challenge were also observed. results: phylogenetic analysis of the complete genomic sequence of yc and the nucleotide sequence of s gene demonstrated that the yc pedv strain was clustered with the pedv epidemic strains, with > % nucleotide identity to these pedv strains. the s gene sequence of yc shared only . % ~ . % identities with classical cv , dr and js strains, with nucleotide insertion in three sites and three nucleotide deletion in one site. the amino acid (aa) sequence of s gene of yc shared only . % ~ . % identities with classical cv , dr and js strains, with aa insertion in two sites and aa deletion in one site. in the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on th day after farrowing of the inactivated yc pedv strain immunized group were significantly higher than the inactivated cv immunized group and the inactivated dr immunized group (p < . ). the traditional inactivated pedv vaccines made from cv or dr could not protect piglets from yc challenge, while inactivated yc could provide piglets with % protection against yc challenge. conclusions: the results showed that, great antigenicity variation had occurred to this yc pedv strain. the yc pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, positive-sense rna virus that is taxonomically classified within the family coronaviridae, genus alphacoronavirus. pedv is the main causative agent of porcine epidemic diarrhea (ped), a devastating enteric disease that is characterized by watery diarrhea, vomiting, dehydration and significant mortality in piglets. approximately to % of pedv-infected piglets die within h of being infected with virulent pedv strains, resulting in tremendous economic losses to the swine industry [ , ] . since december , a large-scale outbreak of diarrhea, characterized by watery stool, dehydration, and vomiting, with to % morbidity and to % mortality in suckling piglets, has been observed in swine farms in china [ , ] . accumulated evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [ , ] . in the present study, a pedv strain, yc , was isolated from intestinal samples of suckling piglets with acute diarrhea in , the evolutionary characteristics and the immune protective efficiency of yc were also determined. in the pedv isolation and propagation experiment, the partial gene of nucleocapsid protein was analyzed by rt-pcr using primers n /n , which amplified an approximately kb nucleocapsid gene fragment present in the isolated yc infected vero cells, but not in the blank control vero cells (fig. a) . the isolated yc pedv strain was detected in the cytoplasm of infected vero cells by an anti-pedv n protein polyclonal antibody. red fluorescence could be observed in the yc strain-infected vero cells (fig. b) . the isolated yc pedv strain was confirmed to be negative for other porcine enteric viruses, such as rotavirus groups a, b and c, tgev, prcv, calicivirus and porcine deltacoronavirus by rt-pcr. the growth kinetics study showed that yc replicated rapidly and efficiently in vero cells, reaching a maximum titer > tcid /ml by hpi (fig. ) . a total of , nucleotides were sequenced in the isolated yc pedv strain, including the polyprotein, s, orf , e, m, and n protein-encoding genes. the sequence of yc was submitted to genbank (accession no. ku ). the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . . phylogenetic analysis demonstrated that the yc pedv strain was clustered with the pedv epidemic strains (g cluster, fig. a and b), with > % nucleotide identity to these strains. the complete genome nucleotide sequence shared . %~ . % identities with classical cv , dr , js , ah-m and sd-m strains (g cluster). the s gene sequence of yc shared only . %~ . % identities with the pedv g cluster, such as cv , dr and js strains, with nucleotide insertion in three sites ( bp, ~ bp, ~ bp) and three nucleotide deletion in one sites ( ~ bp). the amino acid (aa) sequence of s gene of yc shared only . %~ . % identities with classical cv , dr and js strains, with aa insertion in two sites (aa ~ , aa ) and aa deletion in one site (aa ). nucleocapsid protein specific antibodies tests showed that a antibody response was detected in all the inactivated pedv strains immunized groups days after the first immunization (fig. a) . the antibody levels of all the inactivated pedv strains immunized groups maintain high levels at all the testing time points, but the antibody titers of these three groups were not significantly different (p > . ). the results of pedv neutralizing antibodies tests showed that the neutralizing antibody levels of the inactivated pedv strains immunized groups gradually increased until days after the first immunization, and still maintain high level at days after farrowing. the neutralizing antibody titer in sera samples of the yc pedv strain immunized group was significantly higher than the other three groups (p < . , fig. b ). the neutralizing antibody titer in the colostrum and the milk on th day after farrowing of the yc pedv strain immunized group was also significantly higher than the other three groups (p < . , fig. c ). after yc challenge, piglets in group one, group two and group four showed significant acute diarrhea. weight gain was reduced, loss of appetite and mental uneasiness persisted, while piglets in group three, group five and group six showed no obvious diarrhea symptoms. two days after challenge, mortality of group one, group two and group four were %. no mortality or obvious clinical symptoms were observed in group three, group five and group six ( table ). ped can generally be controlled using a vaccine strategy. vaccination with killed or attenuated pedv vaccine has been widely carried out in china and other swine raising countries, where ped usually manifests as a mild and enzootic pattern (lower mortality) some years ago. however, severe acute diarrhea outbreaks associated with high morbidity ( - %) and mortality ( - %) were observed in suckling piglets in most areas of china since december , although most sow herds had previously been vaccinated with traditional inactivated pedv vaccines based on cv or dr . in april , ped was diagnosed in the eastern midwest region of the united states, subsequently, it spread rapidly to neighboring states by june . accumulative evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [ ] [ ] [ ] . the s protein makes up the large surface projections of the virion and plays a pivotal role in determining viral-cellular fusion activity and activating the immune system [ ] [ ] [ ] . the variations in amino acid sequence likely changed the immunogenicity of the s protein and led to immunization failure of current commercial vaccines. in this study, we successfully isolated the yc pedv strain from porcine intestinal samples in dead piglets during outbreaks of acute diarrhea. the s gene nucleotides analysis of yc indicated that it was clustered with the pedv epidemic strains, with nucleotide insertion in three sites and three nucleotide deletion in one site compared to classical pedv vaccine strains cv and dr . the variations of the s gene sequence and deduced amino acid sequence of the yc strain compared to traditional pedv vaccine strains may be the reason why some swine farms had well pedv vaccine immunizations but still had sustained epidemic diarrhea which caused huge economic losses. vaccination is one of the most effective ways in preventing ped infection. immunization of sows with pedv vaccines at - days before production will provide substantial passive immunity to the newborn piglets [ ] . in this study, the nucleocapsid protein specific antibody levels of three different inactivated pedv strains immunized groups gradually increased at all the testing time points. however, the antibody titers of these three groups are not significantly different. immunization with inactivated yc could protect piglets from acute diarrhea against homologous strain challenge. since yc was clustered with the pedv epidemic strains, with > % nucleotide identity to most of these epidemic strains, the inactivated yc may protect other variant strains challenge in some extent. however, it needs further refine animal studies to assess this suppose. the results showed that, great antigenicity variation had occurred to this yc pedv strain under the selective pressure of vaccines. therefore, it is important to investigate pedv variants currently circulating in sow herds to assess their ability to allow for cross-protection against highly virulent pedv strains and prevent pedv epidemics. the results showed that, great antigenicity variation had occurred to this yc pedv strain. the yc pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and c neutralizing antibodies detection in the colostrum and the milk on th day after farrowing. * indicates the neutralizing antibody titer of the yc pedv strain immunized group was significantly higher than the other three groups (p < . ) antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. in may , porcine intestinal tracts, intestinal contents and fecal samples were collected from dead piglets during outbreaks of diarrhea on a breeding farm. this farm keeps more than sows and located in yancheng city within the jiangsu province. clinical signs were characterized by acute vomiting, anorexia, and watery diarrhea, with high mortality in piglets less than days old. the diarrhea outbreaks occurred throughout the year and re-occurred at - weeks interval. virus isolation was performed as described previously [ ] . the rna of the isolated yc pedv strain cultures were extracted using the viral rna mini kit (geneaid biotech, taiwan) according to the manufacturer's instructions. the presence of pedv in the vero cell culture was confirmed by reverse transcription pcr (rt-pcr) with one pairs of primers to amplify approximately kb partial sequence of nucleocapsid protein, n : gcaaacggg tgccattatctc, n : ctagctcacgaacagccac attac. the samples of pedv in the vero cell culture were confirmed to be negative for rotavirus a, b and c, transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), caliciviruses and porcine deltacoronavirus via rt-pcr as previously described [ ] [ ] [ ] [ ] [ ] . the pedv yc strain was then identified with immunofluorescence assay (ifa). briefly, vero cells grown on a -well plate were infected with the pedv yc strain. at h post-infection, cells were washed twice with pbs and fixed with cold methanol for min at − °c. cells were then washed three times with pbst and blocked with % bovine serum albumin (bsa) at °c for h. preparations were incubated for h at °c with mouse anti-pedv nucleocapsid protein polyclonal antibody in dilution buffer ( % bsa in pbst), this mouse anti-pedv nucleocapsid protein polyclonal antibody, prepared by our laboratory, was collected from serum of icr mice immunized with purified prokaryotic expressed n protein. after three washes with pbst, cells were treated with a rhodamineconjugated goat anti-mouse igg (cwbio, china) at a : dilution with pbs for min at °c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z , zeiss, germany). after ten passages on vero cells, the one-step growth curve of yc strain in vero cells was monitored at h interval after infection. based on the sequence of the cv pedv strain (genbank: af . ), seven pairs of oligonucleotide primers ( table ) were designed to amplify the different regions of the yc genome. the pcr products were cloned into the puc vector using clonexpress entry one step cloning kit (vazyme) and sequenced by invitrogen biotechnology (shanghai, china). the ′ and ′ ends of the genome of yc were validated using the rapid amplification of cdna ends (race) cdna amplification kit (clontech, japan). all fragments were sequenced in both directions in triplicate. the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . software. phylogenetic trees were constructed using the maximum likelihood method and supported with a bootstrap test of replicates. genomic sequences of the isolated yc pedv strain were submitted to genbank under accession no. ku . all experimental protocols were approved by the laboratory animal monitoring committee of jiangsu province and performed accordingly. twelve commercial high-health sows (large white) were randomly divided into four groups (table ) after immunization, sera samples were collected from sows at -day intervals until days after immunization ( days after farrowing) for detection of nucleocapsid protein specific antibodies with commercial indirect elisa kits (biovet, canada) and pedv neutralizing antibodies using yc as indicator virus as described previously [ ] . briefly, μl of pedv strain yc ( . × tcid /ml) was added to an equal volume of the sera samples and incubated for h at °c. the mixture was then inoculated to a -well plate containing confluent vero cells. h later, the culture plate was washed with d-hank's three times, and then added μl of dmem containing % fetal bovine serum (fbs). h later, the culture plate was fixed with cold methanol for min at − °c, and incubated with mouse anti-pedv nucleocapsid protein polyclonal antibody for h at °c, and then stained with fitc-labeled rabbit antimouse igg (santa cruz biotechnology). the serum titers were determined as the reciprocal of the last serum dilution at % or greater fluorescent focus reduction in the infected cell cultures under a fluorescent microscope. the colostrum and the milk on th day after farrowing of each sow were also collected and used for the detection of pedv neutralizing antibodies. the average number of sows farrowing was , piglets in each group was coded and chosen randomly, respectively. on th day after farrowing, thirty piglets were chosen (table ), ten from group one (inactivated cv immunized group), ten from group two (inactivated dr immunized group), five from group three (inactivated yc immunized group), and five from group four (saline treated group). these piglets were divided into six groups (table ) , each group were housed in a separate room and were artificial feeding with milk. piglets in group one to group four were challenged orally with . × tcid of yc . group five were challenged orally with . × tcid of cv . group six were challenged orally with . × tcid of dr . piglets were observed daily after challenge about the diarrhea symptoms. all data were analyzed using one-way anova and values of p < . were considered statistically significant. submit your next manuscript to biomed central and we will help you at every step: pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy new variants of porcine epidemic diarrhea virus, china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu complete genome sequence of a chinese virulent porcine epidemic diarrhea virus strain sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/ from the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain propagation of the virus of porcine epidemic diarrhea in cell culture detection and genetic diversity of porcine group a rotaviruses in historic ( ) and recent ( and ) swine fecal samples in ohio: predominance of the g p[ ] genotype in nursing piglets detection of group b and c rotaviruses by polymerase chain reaction detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus pcr-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in us swine construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus (pcv ) in mice this study was supported by program from the jiangsu province science and technology support program (be ), the jiangsu agriculture science and technology innovation fund (cx ( ) the authors declare that they have no competing interests.authors' contributions hxl and hjf designed the study. lc and lg performed the virus isolation. hxl and zm performed the animal test. all authors read and approved the final manuscript. key: cord- -yeaw nr authors: nedjadi, taoufik; el-kafrawy, sherif; sohrab, sayed s.; desprès, philippe; damanhouri, ghazi; azhar, esam title: tackling dengue fever: current status and challenges date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: yeaw nr according to recent statistics, million apparent dengue infections were estimated worldwide in . this figure is by far greater than the who prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas. this article aims at bringing to light the current epidemiological and clinical status of the dengue fever. the relationship between genetic mutations, single nucleotide polymorphism (snp) and the pathophysiology of disease progression will be put into perspective. it will also highlight the recent advances in dengue vaccine development. thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. however, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk. dengue fever is a major cause of illness and death worldwide. the disease is caused by dengue virus which gets transmitted to humans by the bites of infected mosquitoes, aedes (ae.) aegypti and ae. albopictus [ ] . the disease represents a global health issue as it is endemic in around countries, most of which are in tropical and sub-tropical areas. over the last decades, the incidence rate and the geographic distribution of dengue have rapidly increased (almost -fold). data from the world health organization (who) estimates up to million cases of dengue fever each year [ ] . however, a recent published work by bhatt et al. ( ) suggested that the burden of dengue is far more than the who estimation and indicated that million infections of dengue virus could have happened every year [ ] . changes in dengue epidemiology and the increase in incidence rates (with and without co-morbidities) have led the who to propose a new dengue classification system according to disease severity ( fig. ) [ ] . dengue fever is caused by infection with dengue virus (denv). the denv is a vector-borne virus transmitted to humans primarily by bites from two mosquito species, ae. aegypti or ae. albopictus. denv is a single positivestranded rna virus belonging to flavivirus genus of the flaviviridae family and has major serotypes (denv - ) that are antigenically distinct from each other. each denv serotype is phylogenetically distinct suggesting that each serotype could be considered a separate virus [ ] . three dengue serotypes out of four (denv - ) have been found in middle eastern countries including saudi arabia and yemen. interestingly, denv- strain isolated in saudi arabia exhibited a high genetic similarity with denv- strain isolated from asian population, suggesting a widespread of the asian genotype, probably through asian pilgrims [ , ] . a recently published article has unveiled a new serotype , to be added to the existing ones [ ] . this discovery is still controversial and little-known enough to conclude how the th dengue serotype might add to the burden associated with dengue infection. mosquitoes transmit the virus by feeding on blood of infected persons. at first, the virus infects and replicates in the mid-gut epithelium of the mosquito and then spreads to other organs until it reaches the salivary glands after - days where it can be inoculated to another person during subsequent blood meal. vertical transmission of denv in mosquitoes, i.e. from mosquito to larvae has been reported by a number of research groups. in india, angel & joshi ( ) reported the detection of dengue virus by indirect fluorescence antibody test (ifat) in laboratory reared mosquitoes originating from larvae collected from urban and rural areas [ ] . a similar study was conducted in brazil by martins et al. ( ) and confirmed the isolation of denv-type in ae. albopictus larvae and denv-type in ae. aegypti larvae [ ] . similar findings were also reported in mexico [ ] and indonesia [ ] . on the other hand, mother-to-infant transmission of dengue virus via cord blood or breast milk remains controversial [ ] [ ] [ ] . based on the results from several studies, the who has launched a new dengue classification. this classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases [ ] . however, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. the criteria for dengue case classification are presented in fig. . clinically, dengue infection has a broad spectrum of features. the vast majority of cases are asymptomatic and passes unnoticed. typically, the symptoms start to be prominent after an incubation period of - days [ ] . the severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [ ] . predicting the progression of the mild signs to a severe dhf/dss remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms. the early signs of the disease are non-specific. according to the who classification ( ), df is characterized by febrile episode (≥ °c for - days) frequently associated with rash, nausea, vomiting, and headache. although the disease affects people of all ages from infancy through to adulthood [ ] , epidemiological data showed that children tend to tolerate this phase of illness better than adults [ ] . the persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen - days later. laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity [ ] . the emergence of these symptoms is a warning sign for disease progression to severe form (dhf/dss) if therapeutic intervention is not undertaken. at this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area. this form of dengue infection can be attributed to any of the four known serotypes denv - . the likelihood of developing dhf/dss is high in patients who have experienced dengue infection in the past with heterogeneous serotype [ ] . about - % of patients progress to develop a severe dhf/dss which can be fatal unless treated promptly [ ] . this form develops at a late stage of df, where patients may go through defervescence phase characterized by a sudden drop of body's temperature. this phase is also distinguished by severe bleeding, particularly bleeding from the gastrointestinal tract (black, tarry stool), and thrombocytopenia (< , /mm ), which may affect up to % of dhf cases [ ] . interestingly, there was an observed negative correlation between the severity of dhf and the level of platelets in the blood. the exact mechanism of this correlation has yet to be delineated. the drop of platelet counts and the loss of their functionality lead to a vascular fragility increasing the risk of hemorrhage and plasma leakage [ ] . it has been suggested that during acute phase of the infection denv replicates quickly in platelets, as this is very critical for virus survival and dissemination [ , ] . the existence of other symptoms such as retro-orbital pain, maculopapular rash, petechiae, or bleeding from the nose or gums will help making definitive diagnosis for df [ ] . evidence of plasma leakage in various body cavities such as the pleural cavity and the peritoneal cavity, associated with profuse perspiration, adynamia, and sometimes fainting are signs of rapid progression to shock. subsidence in systolic pressure and hypotension may result in profound shock, known as dengue shock syndrome (dss). the duration of dss for a long time might predispose to further complications such as massive bleeding, disseminated intravascular coagulopathy (dic), respiratory failure, multi-organ failure, and infrequently encephalopathy leading to death [ , ] . it has been proposed that case fatality related to dhf may reach % of all cases, however, proper medical care and symptomatic management can reduce mortality rate to less than % [ ] . an early and accurate laboratory diagnosis of dengue infection is of paramount importance in the management of the disease. it has been estimated that the number of misdiagnosed dengue cases could reach a record ratio of % of all cases, mainly due to a large disparity of dengue signs and symptoms which overlap with the symptoms of other viral infections, especially for persons living in or traveling to endemic areas of tropical infectious diseases. dengue fever should be distinguished from other illnesses which share similar symptoms such as chikungunya, mayaro fever, ross river fever, west nile fever, zika fever, yellow fever and viral hemorrhagic fevers [ ] . until the antiviral vaccine becomes available, the prevention of severe cases and cut-down of the economic burden of the disease rely enormously on early and accurate diagnosis. the latter is made possible through the availability of several diagnostic laboratory and virological tests. the onset of later stage symptoms of the illness can be overwhelming and more pathognomonic. nonetheless, based on who classification schemes, the appearance of leukopenia in patients with febrile illness is a major consideration in making diagnosis of dengue infection [ ] . overall, there is an urgent need to reduce dengue morbidity and mortality by improving the diagnosis and molecular analysis of emerging dengue virus. thus far, two diagnostic modalities have been applied to detect the disease at an early stage. the first one is a direct method targeting the acute phase of dengue disease, which is based upon detection of genomic rna by rt-qpcr or soluble ns by antigen capture in blood samples from viremic patients. the second is the indirect method that relies on serological tests to detect denguerelated immunoglobulins par mac-elisa for the capture of specific igm or indirect elisa for the capture of anti-den iggs [ ] [ ] [ ] [ ] [ ] . several risk factors have been associated with dengue infection and its progression to severe dhf/dss forms. recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the dhf progression [ , , ] . hence, an in-depth analysis of genetic variability including polymorphism and mutations could be beneficial in identifying the possible factors and mechanisms of disease development [ ] . the list of host's genetic factors that confer susceptibility or resistance to dengue infection is summarized in table . like most arboviruses, denv infect different organs of the mosquito, including the salivary glands and the central nervous system. mosquito infection elicit behavioral changes including increase of the probing time which lead to host interruption that might lead to wider spread of the virus [ ] . it has been demonstrated that denv infection induced the expression of cathepsin-b, a putative cystatin, and a hypothetical ankyrin repeat-containing protein genes [ ] . the latter could alter the efficiency of virus replication in the salivary gland. this study has shown that modulation of obp and obp genes expression as well as denv infection-responsive odorant-binding protein genes increase the time length for initiation of probing before a successful blood meal, resulting in changes in the host seeking behavior of the mosquito. comparative analysis of the salivary gland transcriptomes of native and denv-infected ae. aegypti identified a number of differentially expressed genes related to sugar/protein digestion enzymes, immunity related genes and blood meal acquisition enzymes that might have an impact on the efficiency of viral replication or mosquito feeding behavior. this study showed that denv infection alter the expression of key host-seeking genes in the mosquito's main olfactory organs and the antennae [ ] . recent updates have indicated that resistance of ae. aegypti to conventional insecticides is related to different mechanisms, one of which is associated with genetic abnormalities within the vector's genome. single point mutation in the voltage-gated sodium channel gene at position (f c) resulting in phenylalanine to cysteine substitution in ae. aegypti confers resistance to permethrin. this mutation is widespread in this vector in southeast asia and latin america [ , ] . it has also been reported that a single amino acid substitution valine to glycine at position in domain ii, segment of the voltage-gated sodium channel gene was associated with less sensibility of ae. aegypti to deltamethrin in thailand [ ] . numerous multi-disciplinary studies confirmed that race, young age, virus strain, female sex and high body-mass index correlate well with increased burden of dengue [ , ] . thus, a closer consideration of human genes regulating the severity of dengue infection, especially genes associated with the immune response, might help in controlling disease spread and improve the acute symptoms of the infection. a number of studies have investigated the relationship between the host genetic polymorphisms and denv infection ( table ) . a single nucleotide polymorphism (snp) in the promoter of cd /dc-sign was associated to increased risk of developing dengue fever [ ] . association studies have successfully identified a link between polymorphisms in the human major-histocompatibility-complex (hla) class i/ii genes and non-hla host genetic factors and severity of dengue disease [ ] [ ] [ ] . polymorphisms of the tap and tap genes could be directly associated with the risk of developing dengue disease among the primary-infected individuals [ ] . both tap and tap are located within the mhc class ii region and homozygosity of the tap at position and and for tap at position , respectively, was found to protect against developing severe forms of dengue [ ] . in an independent study [ ] , the authors showed that single nucleotide polymorphism of the oligoadenylate synthetase genes (oas , and ), of the oas/rnase l antiviral immune system, enhance susceptibility to clinical outcomes of dengue infection. an association between the severity of the disease and other genes including human leukocyte antigen class i and class ii genes, tumor necrosis factor-alpha, fcgriia, vitamin d receptor, transporters associated with antigen presentation, and jak has also been proposed [ ] . the importance of vit-d in denv pathogenesis was concluded from newly-gathered data showing that vit-d impairs denv replication and polymorphism of vit-d gene increases the expression of both cd /dc-sign and fcgriia receptors that enhance denv entry in the target cells [ , ] . in another study [ ] , the authors have successfully applied genome-wide association study (gwas) approach to identify loci that confer susceptibility to severe forms of dengue disease. the investigators used samples from children affected with severe dengue infection against population control cases in vietnam. the data showed that snps at two loci, micb and plce , significantly increased the likelihood of developing dss in children. this finding was further validated in an independent cohort of cases and controls [ ] . a snp in the micb gene coding for the mhc class i polypeptide-related sequence b, an inducible activating ligand for the nkg d type ii receptor of immune cells could alter the protective role of natural killer and cd + t cells in the host responsiveness to denv at the early stage of infection [ , ] . on the other hand, plce plays a primordial role in maintaining intact vascular endothelial cell barrier function, hence, polymorphism of the plce gene may lead to blood vessels leakage and circulatory hypovolemia during dss [ ] . other host candidate genes have also been associated with early onset dengue disease. among these genes, there were receptors/attachment factors for denv linked to immune system and inflammatory response. the chemokines cxcl , cxcl and its respective chemokine receptor cxcr were reported as biomarkers for severe form of dengue infection [ ] . these results are in agreement with recent emerging data indicating strong association between cxcl , cxcl and cxcr and vascular permeability [ ] . the three genes are components of the nf-kb pathway and are involved in the pathogenesis of sars and west nile virus encephalitis [ , ] . [ ] . this process depends enormously on vector-derived salivary factors inoculated on the skin cells [ ] . till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, dna vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones ( table ) . currently, only one tetravalent vaccine against dengue virus, developed by sanofi-pasteur (france) has reached phase iii clinical trial and is expected to be launched in . this vaccine is based on the production of four chimeric live dengue-yellow fever viruses in which the yellow fever (yf) d vaccine sequences encoding the envelope proteins prm and e genes were substituted by the prm and e genes from dv of serotype , , , or in a molecular clone of yf- d [ ] . this vaccine was produced and tested over people using four dengue virus isolates from indonesia and thailand. this candidate vaccine was found to be attenuated and stable in animal models with respect to plaque size and yellow fever virus neurotropism [ ] . results of the clinical trials showed no adverse effects except moderate injection site pain, headache, and myalgia. another randomized, controlled trial was launched using a total of thai school children to investigate the efficacy of a recombinant, tetravalent vaccine for dengue virus and only dengue cases were reported [ ] . phase i trial of the vaccine in the philippines showed that the seropositivity increased gradually ( , & %) after - vaccinations against all four serotypes as compared to control group. the most promising results were observed in children - years old who exhibited high levels of reactivity of , , , % for denv - ; respectively [ ] . another placebo-controlled trial was conducted on , children from vietnam (vaccine, n = vs placebo, n = ) to determine the clinical efficacy and safety of cyd-tdv. the results demonstrated virologically-confirmed cases in % of the vaccine group as compared to the control group ( %). the efficacy was achieved in up to . % ( % ci . - . ). these findings indicated that the vaccine is highly efficacious with good safety profile when three injections were given to children with age group - years at , and months intervals [ ] . the data emerging from another randomized phase ii trial in india indicated that the vaccine has no serious adverse events and the immunogenicity and safety of cyd-tdv were satisfactory [ ] . a pilot study carried out in five latin american countries where more than , children aged - were recruited to receive either the cyd-tdv vaccine or placebo. the results on efficacy ( . %) and safety profiles were consistent with the previous findings [ , ] . interestingly, the vaccine efficacy ( . %) against hospitalization for dengue was promising and represented a step forward to developing an effective dengue vaccine [ ] . other candidate dengue vaccines have been developed in usa by the johns hopkins university and national institute of allergy and infectious diseases (niaid) and have reached advanced clinical trials [ ] . four liveattenuated denv/delta- were generated each containing nucleotides deletion of the '-untranslated region of genomic rna (delta- ). these vaccines efficiently impaired viral growth in human liver carcinoma cells [ ] . to improve the attenuation of denv- /delta- and denv- /delta- , chimeric denv were developed by substitution of the prm-e gene region of denv- / delta- virus with the prm-e genes of denv- and denv- [ , ] . the results from phase i clinical trial showed that all four live-attenuated denv/ delta- are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [ , ] . dengue virus serotype- antigen was expressed in a vector based on pediatric live-attenuated schwarz measles vaccine (mv) by using the envelope domain iii (ediii) fused with the ectodomain of the membrane protein (ectom). after immunization, long-term production of denv- serotype-specific neutralizing antibodies was observed in measles virus susceptible mice [ ] . a new strategy was evaluated based on single minimal tetravalent denv antigen expression using viral vector derived from pediatric live-attenuated measles vaccine (mv). a recombinant mv vaccine construct was developed using envelope domain iii (ediii) and ectodomain of the membrane protein. the neutralizing antibodies were induced against all four serotypes of dengue virus after two injections in mice susceptible to mv infection. a strong memory neutralizing response was observed against all four serotypes in immunized mice after inoculation with live denv from each serotype [ ] . a naked dna-based candidate vaccine against denv has been developed by the naval medical research center [ , , ] . the genes encoding prm and e of denv were cloned into a shuttle vector under the transcriptional control of human cytomegalovirus (cmv) promoter. the results of phase i clinical trial showed no adverse effects except mild injection site pain, swelling, and fatigue. after second dose, strong igm and igg antibody response was observed which favors the safety profile of this vaccine. to get a better immunogenicity profile, a vaccine based on lipid adjuvant vaxfectin (vical incorporated, san diego, usa), was developed and the results demonstrated good protection profile against denv compared to dna alone [ ] . based on this technology, different groups have developed other candidate vaccines and achieved good protection in mouse models using envelope glycoproteins prm and e, the non-structural protein ns and the helicase/protease ns as vaccine antigens [ ] [ ] [ ] . the first purified inactivated vaccine was developed with aluminum hydroxide (alum) adjuvant and tested in mice and rhesus macaques in the mid- s, by walter reed army institute of research against dengue serotype and good virus protection was reported after two doses [ , ] . using similar technology, second generation japanese encephalitis (je) piv vaccine was developed [ , ] . currently, a new je vaccine (ixiaro; novartis vaccines) has been approved for use in many countries, including the usa [ ] . another dengue vaccine (dengue piv), recombinant subunit dengue e glycoprotein antigen (r e) was also developed and has entered phase i clinical trial [ ] [ ] [ ] . the centers for disease control and prevention (usa) have also developed a live-attenuated vaccine named denvax, which was found to be highly immunogenic in both children and adults and has currently entered phase i clinical trial in the united states [ , ] . recently, a novel third generation approach is being used to develop a vaccine containing recombinant subunit e domain iii (ed ) and the results of laboratory tests have shown the development of potent neutralizing antibodies in a mouse model [ ] [ ] [ ] . using the same technology, a tetravalent vaccine was developed and expressed in pichia pastoris by splicing and using flexible pentaglycyl linkers of the four ediii. the observed results showed that this antigen elicit specific antibodies against all four denv serotypes in balb/c mice [ ] . animal models are very useful for vaccine test development. the lack of animal models significantly hampered the development and efficacy testing of dengue vaccine. currently only rhesus macaques and aotus monkeys are being used for testing the vaccine before clinical trials are initiated [ ] . the d me vaccine was evaluated in both aotus monkeys and rhesus monkeys, and found to be immunogenic with - % protection against dengue infection [ , ] . porter et al. ( ) demonstrated that injection of non-human primate with three doses on day , and , with tetravalent dengue dna vaccine vaxfectin-adjuvanted, was more efficient against live dengue- virus compared to control animals. this finding support initiation of vaxfectin-adjuvanted phase i clinical trial [ ] . successful induction of immune response was obtained in mice and rhesus monkeys to the vaccines developed using dengue prm-e, dengue prm-e-nonstructural (ns) , and dengue ns antigens, and piv adjuvanted with alum [ , ] . centers for disease control and prevention (fort collins, co), hawaii biotech, and simmons developed different vaccines that showed good immunogenicity in animal models [ ] . similarly, the psoralen/uv inactivation dengue vaccine was found to be more immunogenic and protective against dengue serotype virus in aotus monkeys [ ] . thus far, there are no antiviral drugs available to treat dengue fever; therefore the community will continue to depend on the control of the mosquito vector as the main route to prevent the spread of disease. alternative approaches have been utilized against flaviviruses by targeting and inhibiting virus entry and the essential elements used in virus replication, nonstructural proteins, rna polymerase, and proteases. the most important target elements include ns helicase nucleoside triphosphatase (ntpase/ rna ' triphosphatase (rtpase), ns methyl transferase/ rna-dependent rna polymerase, and ns /ns b protease [ ] [ ] [ ] . rna interference (rnai) technology is also being used to impair virus replication against respiratory syncytial virus, hepatitis viruses, influenza virus, poliovirus and hiv [ , ] . low molecular weight phenolic compounds such as flavonoids and phytochemicals isolated from plants were previously tested and are being used for anti-dengue therapy [ , ] . an anti-viral inhibitory effect ranging from - % against denv replication was observed when methanolic extracts of momordica charantia and andrographis paniculata were used in cultured primate cells [ ] . several attempts have been made in the past to tackle dengue through elimination of ae. aegypti. the most successful experiences were related to vector control programs adopted in cuba and singapore. the programs were based on intensive insecticidal treatment and reduction of the availability of aedes larval habitats [ , ] . unfortunately, lack of sustainability of these stringent measures led to reappearance of dengue outbreaks. recently, a novel form of biological control of dengue transmission has been developed and is currently being applied. this is based on the development of genetically modified (gm) mosquitoes infected with a bacterium known as wolbachia to combat dengue infection. this bacterium blocks replication of the virus inside the mosquito and prevents its transmission to humans [ ] . in , million gm male mosquitoes were released in the wild to decrease the number of aedes mosquitoes and reduce the rate of dengue transmission. a closer monitoring of the insects revealed that over % of the eggs were wolbachia-positive which indicated that gmmosquitoes were overriding wild-mosquitoes resulting in decreased virus transmission [ ] . in an initiative to eradicate dengue fever, scientists from australia, are leading eliminate dengue (ed) program which involves community engagement as a key component in this program. since the program kicked off in , millions of wolbachia mosquitoes were released across the north queensland city-australia. based on the promising results obtained from local trial, eliminate dengue became an international research program across countries affected by dengue including australia, vietnam, indonesia, brazil and colombia [ , ] . dengue infection can be prevented by alternative approaches. the first one includes blocking virus entry into cells which is mediated by the viral envelope glycoprotein e via receptor-mediated endocytosis [ ] . dendritic cells, monocytes, and macrophages are the main targets of denv infectious entry. the second approach involves blocking virus attachment to specific cellular receptors expressed on immune cells, liver cells, and endothelial cells. small molecules and peptides targeting the hydrophobic pocket of the envelope e glycoprotein are characterized as inhibitors of virus entry. nicholson et al. ( ) explored the inhibitory effects of dn and oan , peptide entry inhibitors. the authors demonstrated that dn and oan can effectively block antibody dependent enhancement (ade) in-vitro suggesting that entry inhibitors are potential candidates to prevent development of dhf/ dss [ ] . two other compounds have also been shown to qualify as potent inhibitors of dengue virus infection are imino-sugars deoxynojirimycin and castanospermine [ ] . these compounds are natural alkaloids derived from the black bean and act as inhibitors against all dengue serotypes by disrupting the folding pathways of the envelope glycoproteins prm and e [ ] . various types of carbohydrate-binding agents, isolated from different organisms, have been shown to have antiviral activities. three plant lectins, hippeastrum hybrid agglutinin, galanthus nivalis agglutinin and urtica dioica agglutinin isolated from amaryllis, snowdrop and stinging nettle respectively were found to be potent inhibitors of denv- infection by inhibiting viral replication [ ] . heparan sulfate (hs) is a putative receptor for denv which interacts with domain iii of the e-protein. virus entry can be blocked by targeting the e-protein-hs interaction with soluble gags and other highly charged hs [ ] . fucoidan was isolated from marine algae and showed antiviral activity against denv- in bhk cells [ ] . similarly, carrageenan and dl galactan, sulfated polysaccharides from red seaweeds, exhibited strong antiviral activity against denv- and denv- but a very weak activity against denv- and denv- . furthermore, two α-d-glucans were isolated from a chinese herb and demonstrated high anti-denv- activities in bhk cells [ , ] . dengue fever represents a real economic burden especially in affected countries. extensive efforts are needed to tackle disease spread and reduce the mortality rates and the associated healthcare cost. there is a need for more scientific research which we believe is a key route to provide further insight in the pathogenesis of dengue infection and help understanding the underlying molecular mechanisms associated with progression to the severe forms of the disease (dhf/dss). this will be a step forward to develop an adequate preventive vaccine and effective treatment. the authors disclose that there is no conflict of interest. authors' contributions tn participated in the review design, coordination and helped to draft the manuscript. sk and ss participated in the review design and helped to draft the manuscript. pd participated in the article revision. gd and ea participated in the review design. read and approved the final manuscript. this project is funded by the king abdulaziz city for science and technology (kacst) under grant number ( ‫ﺍ‬ ‫ﺕ‬ - - ). the authors are also grateful to the diagnosis of dengue: an update guidelines for diagnosis, treatment prevention and control the global distribution and burden of dengue dengue viral infections outbreak of viral hemorrhagic fever caused by 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chimeric dengue- pdk- -based tetravalent vaccine for protection against dengue fever immunogenicity and efficacy of chimeric dengue vaccine (denvax) formulations in interferon-deficient ag mice next-generation dengue vaccines: novel strategies currently under development next generation dengue vaccines: a review of candidates in preclinical development a tetravalent recombinant dengue domain iii protein vaccine stimulates neutralizing and enhancing antibodies in mice an envelope domain iii-based chimeric antigen produced in pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes a dengue virus serotype- dna vaccine induces virus neutralizing antibodies and provides protection from viral challenge in aotus monkeys immunogenicity of dengue virus type dna vaccines expressing truncated and full length envelope protein protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy 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dengue. males with offspring-killing genes are replacing wild insects, say researchers successful establishment of wolbachia in aedes populations to suppress dengue transmission designing a community engagement framework for a new dengue control method: a case study from central vietnam dengue virus entry as target for antiviral therapy viral entry inhibitors block dengue antibody-dependent enhancement in vitro α-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo antiviral activity of carbohydrate binding agents and the role of dc-sign in dengue virus infection antiviral effect of the heparan sulfate mimetic, pi- , against dengue and encephalitic flaviviruses structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga structure elucidation and sulfated derivatives preparation of two α-dglucans from gastrodia elata bl. and their anti-dengue virus bioactivities submit your next manuscript to biomed central and we will help you at every step: key: cord- -ps rzjve authors: zhao, fu-rong; liu, chun-guo; yin, xin; zhou, dong-hui; wei, ping; chang, hui-yun title: serological report of pandemic (h n ) infection among cats in northeastern china in - and - date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: ps rzjve background: influenza a virus has a wide range of hosts. it has not only infected human, but also been reported interspecies transmission from humans to other animals, such as pigs, poultry, dogs and cats. however, prevalence of a (h n ) pdm influenza virus infections in cats in northeastern china is unknown. therefore, the prevalence of a (h n ) pdm influenza virus infections was performed among cats in northeastern china in this study. findings: of all samples in this study, the overall seroprevalence of pandemic (h n ) infection in cats was % ( / ). it also showed a higher prevalence rate of pandemic(h n ) infection in pet cats ( . %) than roaming cats ( %) based on nt. in addition, the results also showed a trend of difference in term of species of cats and it was statistically significant. conclusions: this is the first survey on the seroprevalence of pandemic (h n ) infection among cats in northeastern china. this study has observed a relatively high seroprevalence of pandemic (h n ) among different cat populations in northeastern china, similar seroprevalence studies should be conducted elsewhere. influenza a virus has a wide range of hosts. often the susceptibility of the species is dependent upon the characteristics of the virus and host. numerous subtypes of influenza a viruses, including influenza a pandemic h n virus, have been shown to cross-species transmission. since , a novel influenza a virus (h n ), now called a (h n ) pdm influenza virus, has caused human influenza outbreaks in north america [ ] and a worldwide pandemic [ ] [ ] [ ] . to date, it has not only infected human, but also been reported interspecies transmission from humans to other animals, such as pigs, poultry, dogs [ ] [ ] [ ] . recently, the reports have shown that cats can also infected a (h n ) pdm influenza virus [ , ] . due to frequent cohabitation and close contacts with humans and other animals, cats are uniquely positioned to serve as reservoirs for influenza virus infection both within a household and within the larger farm or rural environment in china [ , ] . however, prevalence of a (h n ) pdm influenza virus infection in cats in northeastern china is unknown. therefore, the prevalence of a (h n ) pdm influenza virus infections was performed among cats in northeastern china in this study. a total of feline blood samples were collected from different pet hospitals and four small animal shelters around northeastern china, from february to march . the geographical and prevalent distribution of the samples has been concerned. haerbin, changchun and shenyang were selected since they are the most densely populated area of commerce in northeastern china. dalian was also included as it is the trade zone with large-scale breeding of poultry and pigs in northeastern china. the geographical location of serum samples of collection in northeastern china was displayed, please see the figure . blood samples from pet cats in hospitals and blood samples from roaming cats were obtained. in each city, we selected the single largest small shelter. these serum samples were separated by centrifugation at , rpm for min, and supernatants were transferred to a new eppendorf tubes and stored at− °c until tested for antibodies against influenza a virus [ ] . additionally, in order to have a timely data for pandemic (h n ) prevalence in northeastern china, blood samples were retrospectively analyzed from pet dogs and pet cats in harbin in . all samples were tested by hemagglutination inhibition (hi) and neutralization (nt) assay, according to the recommended procedures as previously reported [ , ] . hi titer ≥ and nt titer ≥ are considered as positive and indicate previous infection [ ] . influenza virus used in this study was a/california/ / (h n pdm ) [pandemic (h n ) virus]. we additionally studied the sera for hi antibodies against three other viruses: a human seasonal h n influenza virus a/brisbane/ / (h n ) and a/ canine/guangdong/ / (h n ), a recently circulating h n canine influenza virus (civ) in dogs in china. the comparison of categorical variables between cat samples was performed with chi-square test where appropriate. statistical significance was defined as p < . . the data was analyzed with sas software, version . [ ] . a total of serum samples were examined by nt and hi for pandemic (h n ) antibodies. the serological screening revealed % pandemic (h n ) infection in cats in northeastern china based on nt. it also showed a higher prevalence rate of pandemic (h n ) infection in pet cats ( . %) than roaming cats ( %) based on nt (p = . , table ). the results from hi also showed a trend of difference in term of species of cats and it was statistically significant (p = . ). the prevalence of the infection also showed a geographical difference in roaming cats as prevalent in harbin and changchun ( . % and . %) and absent in shenyang and dalian (table ). in addition, to rule out non-specific cross-reactivity, serum samples were titrated against seasonal influenza viruses (h n ). only twenty-four samples had a hi titer of : against h n (table ) . only ten of these forty seasonal influenza positive-samples were also hi and nt positive for a/california/ / (h n pdm ). a total of ( . %) sera were positive by hi assay against h n civ (table ) . positive. in another conducting the seroprevalence of antibodies against (h n ) pdm among cats in small cities of southern china was only . % in [ ] . our increased antibody prevalence might be explained a number of ways. perhaps cats were at a higher probability of infection in northeastern china, due to they exposures in dense populations of humans with high influenza a (h n ) pdm attack rates. the difference might also be explained by the one year temporal difference between cats sampled in southern china in that the northeastern china cats had more years to acquire influenza a (h n ) pdm virus infection. additionally, the prevalence of seropositive pandemic (h n ) in male cats versus female cats suggests that the male cats may be more susceptible (p < . ) to the pandemic (h n ) infections (table ) . we hypothesize that relatively high a (h n ) pdm transmission may have occurred between humans and cats during the period of virus infection in the human population. this hypothesis is supported by our observation that pet cats were more likely to have evidence of previous infection with a (h n ) pdm that were roaming cats ( . % vs %, p = . ) and also suggests a likely transmission between infected owners and their pets by close contact. serological evidence of a (h n ) pdm in domestic cats has been reported in the past. in a sero-survey conducted in italy in , a contrary low prevalence had been observed among dogs, while no cats were reported to have antibodies against a(h n )pdm in the screen [ ] . a similar high prevalence of . % and . % were recorded in a population of cats in the united states, but the study sample comprised animals with a history of respiratory disease [ ] . we hypothesized the sustained transmission of the influenza a (h n ) pdm virus in the human population in our study area. in addition, it should be noted that samples from the two small animal shelters in harbin and changchun had exposure to pandemic (h n ) before sample collection. the higher prevalence of seropositive pandemic a (h n ) pmd among harbin and changchun cats versus shenyang and dalian is unexplained. since cats may be exposed to different influenza virus subtypes, including human-avian and avian-origin influenza viruses, their potential role in the epidemiology of influenza prevention: swine influenza a (h n ) infection in two children-southern california influenza pandemics of the th century organization wh: influenza a (h n )-update organization wh: world now at the start of influenza pandemic p: influenza a pandemic (h n ) virus infection in domestic cat avian-origin h n canine influenza virus circulating in farmed dogs in guangdong dog infected with h n ; study shows virus non pork serologic evidence of pandemic (h n ) infection in dogs seroepidemiological evidence of avian influenza a virus transmission to pigs in southern china pandemic and seasonal human influenza virus infections in domestic cats: prevalence, association with respiratory disease, and seasonality patterns serologic evidence of pandemic influenza virus h n infection in cats in china a serological survey of canine respiratory coronavirus and canine influenza virus in korean dogs the seroprevalence of pandemic influenza h n ( ) virus in china serologic survey of the pandemic h n virus in guangdong province, china: a cross sectional study seroepidemiological study of influenza a (h n ) pdm virus following the - wave in shandong province serological report of pandemic (h n ) infection among cats in northeastern china in - and - the authors declare that they have no competing interests. key: cord- -q jnhss authors: bartel, sebastian; doellinger, joerg; darsow, kai; bourquain, daniel; buchholz, rainer; nitsche, andreas; lange, harald a title: proteome analysis of vaccinia virus ihd-w-infected hek cells with -dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: q jnhss background: despite the successful eradication of smallpox by the who-led vaccination programme, pox virus infections remain a considerable health threat. the possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. in this report the proteome of hek cells infected with vaccinia virus strain ihd-w was analyzed by -dimensional gel electrophoresis and maldi-psd-tof ms in a bottom-up approach. results: the cellular and viral proteomes of vacv ihd-w infected hek cells, uv-inactivated vacv ihd-w-treated as well as non-infected cells were compared. derivatization of peptides with -sulfophenyl isothiocyanate (spitc) carried out on ziptipμ-c columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the psd spectra. the expression of more than human proteins was modulated by the viral infection. effects of uv-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. these effects might therefore be attributed to virus entry and virion proteins. however, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. conclusions: the proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. the confidence of protein identifications was clearly improved by the peptides' derivatization with spitc on a solid phase support. some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis. despite remarkable progress in the control and treatment of infectious diseases, the problem of emerging and re-emerging pathogens is likely to be one of the main issues of medical science and public health in the twenty-first century [ ] . in this respect viral diseases are of particular concern, because advances in the field of antiviral drugs have lagged behind those regarding bactericidal drugs and antibiotics. it was shown by the emergence of the severe acute respiratory syndrome (sars) that new members of neglected virus families can cross into humans from unsuspected reservoirs, making rapid advances in our understanding of virushost dynamics necessary [ ] . in this regard poxviruses are of particular importance. despite the success of the who-led smallpox eradication programme, other related human-pathogenic poxviruses remain a considerable threat. in particular, the outbreak of human monkeypox virus in the united states of america pointed out the imminence posed by zoonotic pox infections originating from animal-borne viruses [ ] . moreover, an increasing number of human cowpox virus infections, especially affecting younger people lacking smallpox vaccination, have been reported in europe in recent years [ ] . in addition to the threat of zoonoses, the relevance of a deeper understanding of the interactions of poxviruses with their host cells is pointed out by considering the classification of smallpox as a category a bioterrorism agent by the centers for disease control and prevention (cdc), especially since no acceptable treatment is available and the immunity in the population is declining [ ] . in contrast to many other viruses, the ability of poxviruses to productively infect a given cell is not determined at the level of specific host receptors, but is regulated downstream of virus adsorption to the cell surface and virus entry by intracellular processes which for the most part are unexplored [ ] [ ] [ ] [ ] [ ] . since the permissiveness of orthopoxviruses is therefore dependent on the hosts proteome set, the identification of cellular proteins affected by the protein products of the so-called viral host-range genes with proteomic approaches will be a starting point for further functional protein analysis. the characterization of the vaccinia virus (vacv) virions' proteome has enlightened the protein equipment of the virus at the entry stage of infection [ ] . furthermore, virus-host dynamics have been analysed by yeast two-hybrid (y h) studies to identify interaction partners [ ] . no closer look at the dynamic changes of the human proteome during a vacv infection has been taken yet on a global scale. in this report an infection of human embryonic kidney (hek) cells with well-studied vacv was chosen as a model system. uninfected hek cells serve as a control, while vacv-infected hek cells inactivated with uv enable the study of the influence of the virions' proteins on the cellular proteome. the investigation of infection effects on the expression of the cellular proteome was carried out in the late phase of virus replication. an offline-coupling of two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) and matrix assisted laser desorption/ionisation time of flight mass spectrometry (maldi-tof ms) was used in a bottom-up approach. in-gel trypsinated proteins from relevant spots of the -d gels are unambiguously identified via mascot database search of the peptide mass fingerprint (pmf) recorded in positive ion reflectron mode of the maldi-tof ms. identification rate was improved by obtaining additional sequence data of selected peptides in post source decay (psd) mode, whereas a considerably advanced data quality was achieved by n-terminally derivatising the peptides on a ziptipμ-c solid phase support with -sulfophenyl isothiocyanate (spitc) prior to ms measurement. psd spectra of spitc derivatized peptides improve protein identification efficiency improved protein identification by combined mascot search of pmf and psd spectra is shown exemplarily in figure for the spot c- , human prohibitin (phb). database search of the underivatised pmf has not led to unambiguous protein identification. the best hits held protein scores between and at a threshold value for significance of . among these, prohibitin is ranked third with a score of . after spitc derivatization, the ten most intensive m/z values of the derivatised pmf were selected by an ion gate for psd fragmentation. the amino acid sequence of selected peptides can be distinguished considering the series of y-fragments. for the peptide of m/z . the sequence fdagelitqr (figure c ), for the peptide of m/z . the sequence dlqnvnitlr (data not shown) and for the peptide of m/z . the sequence iftsi[ge]dyder (data not shown) was confirmed. sequencing enables the mapping of a peptide to a certain protein within the confidence interval of its m/z value and increases its mascot score. if the sequenced peptides do not represent homologue sections in the human proteome even one peptide sequence can be significant. in case of prohibitin each of the three peptides alone was sufficient for unambiguous protein identification. the combined search of pmf and psd spectra of spitc-derivatised peptides identifies spot c- as being human phb with a mascot ion score of at a threshold for significance of . the peptide mass fingerprint of tryptic digested protein standard serum albumin (bovine) before and after spitc derivatization is shown in figure . the intensity is normalised on m/z value of . of underivatised pmf spectra. the spitc derivatization introduces a mass shift of da to the peptides and has strongly decreased their intensities. many peptides could no longer be detected after derivatization, which negatively affects the sensitivity of protein identification. the underivatised protein standard was unambiguously identified with a mascotscore of and a significant threshold of , but could no longer be identified from the pmf after spitc derivatization. the influence of the n-terminal sulfonation on the quality of psd spectra is shown exemplarily in figure c and d for the m/z value of . of pmol tryptic digested bsa. the intensity is normalised on the precursor ion of the underivatised psd spectra. despite the reduction of the precursor ions' intensity of %, the signal to noise (s/n) ratio of the peaks in the psd spectra as well as the frequency of fragmentation were clearly increased by the n-terminal sulfonation. the improved coverage of the precursor ion y-series raised the mascot ion score of the peptide with the mass of . da from to . the sulfonation of primary amines was done in aqueous solution at slightly alkaline ph value (ph = . ). purification of peptides was carried out with ziptipμ-c columns according to the principle of reversed phase chromatography. generally, spitc derivatization of peptides can be performed in solution prior to column binding as well as solid phase supported. both sulfonation methods were modified compared to the literature with respect to signal intensity in order to enable protein identification with maximum sensitivity [ ] [ ] . exemplarily, the resulting pmf spectra for in-solution trypsinated bsa has been compared with the solid phase supported approach concerning signal intensity, which is shown in figure e /f. the signal to noise ratios (s/n) of m/z values used for identification were about to times higher for peptides derivatised on solid phase support than for the ones sulfonated in solution. the overall signal of pmf spectra in the mass range between and m/z is about times larger for the standard derivatised on c columns. the improved s/n ratio and the increased signal intensity of the solid phase supported approach in psd mode enlarges the coverage of the precursor ions' y-series and simplifies the automation of peak picking. the mascot score of the database search of ten accumulated psd spectra was for the solid phase supported approach and for in solution sulfonation at a threshold value for significance of . protein identification via maldi-psd-tof ms spectra of on solid phase support spitc derivatized peptides the recording of psd spectra from m/z values of peptide mass fingerprints provides information about the peptides' primary structure and enables the unambiguous identification of proteins from few peptides with significant mascot ion scores by an analysis of fragmentation patterns. the difficulties of protein identification with maldi-psd-tof ms spectra are low s/n ratios of fragments, incomplete sequence coverage and complex fragmentation patterns as a cause of random charge stabilisation. the protonation of peptides during the maldi process is mediated by carboxyl groups of the matrix and mainly stabilised at primary amine groups of the n-termini and the lysine residues. the stabilised charge induces different fragmentations of the same ion. the derivatization of peptides with spitc binds a negatively charged sulfonic acid to the peptides' n-terminus [ ] . this negative charge neutralises the protonation of c-terminal fragments which are no longer detectable in mass spectrometry. primarily, the psd spectra of spitc-derivatised peptides show fragments of the y-series, whose appearance among the n-terminal fragments is promoted by the large electron density around the peptide bond. the derivatization with spitc enables the detection of complete y-series with good s/n ratios and offers the elucidation of the peptides' primary sequence. the unambiguous mapping of peptides with completely known primary sequences to proteins registered in databases is remarkably simplified and is expressed in increased mascot scores [ ] . however, the application of spitc derivatization for protein identification from complex mixtures is limited by the reduction of the peptides' signal intensities following n-terminal sulfonation. this degradation of sensitivity could be decreased by an optimization of the reaction conditions. for increased sensitivity the n-terminal sulfonation of peptides bound on c solid phase support is preferable compared to an in solution approach since signal intensity is increased and quality of psd spectra is raised. the comparison of the proteome analysis of vacv ihd-w-infected hek cells with non-infected control cells h post infection has enabled the identification of human proteins, whose expression has been regulated by infection, as well as of viral proteins. since the genome of vacv ihd-w is not available, the identification of viral proteins occurred via sequence homologies of the vacv strains western reserve and copenhagen. afterwards peptide sequences were derived from spectra of spots representing proteins regulated in their expression, whose database search gained no result. single-base substitutions among different vacv strains, which led to amino acid replacement, might have resulted in different peptide mass fingerprints. psd spectra are in correlation to the confidence interval of the m/z values of the precursor ion. therefore, protein identification via mascot search is disabled by mass shifts of precursor ions resulting from an amino acid replacement. de novo sequencing of viral proteins could not be assured because of the polyproteonality of d-gel spots. the unambiguously identified differences in the expression profile of hek cells resulting from vacv ihd-w infection are discussed in the following sections. the infection of hek cells with active and inactivated vaccinia virus ihd-w significantly affects the cells' energy metabolism. the expression of fructosebisphosphate aldolase a (a- ), which acts as a central enzyme in the glycolysis by catalysing the retro-aldol cleavage of fructose- , -bisphosphate into dihydroxyacetone phosphate and glycerinaldehyde, is increased by both types of infection [ ] . also the enhanced expression of the enzyme malate dehydrogenase (a- ) is directly associated with a boost in the energy metabolism, which serves the viruses' need for energy for dna replication. this enzyme is an integral part of the citric acid cycle by catalysing the oxidation of malate to fumarate [ ] . the resulting reduction equivalent nadh/h + transfers electrons to the electron transport chain which builds up a proton gradient at the inner mitochondrial membrane, resulting in atp-synthesis catalysed by the h + -transporting two-sector atpase (a- , a- ) [ ] . a modification of the α-chain precursor of the h + -transporting two-sector atpase (a- , a- ), resulting in spot migration, is triggered by both types of infection and has been detected as well as an overexpression of the mtdna stabilising atpase family aaa domain containing a (a- ) protein, whose existence has just been evidenced at transcript level [ ] . another energy metabolism-related protein whose expression is up-regulated by the vacv ihd-w infection is the bsubunit of the electron transfer flavoprotein (a- ) which acts as a specific electron acceptor for several dehydrogenases, e. g. malate dehydrogenase (a- ), and transfers them to the respiratory chain [ ] . all changes detected in the human proteome profile caused by the infection which are related to energy metabolism indicate an enhancement of the metabolic rate of the glycolysis as well as of the oxidative phosphorylation in order to fulfil the viruses' energy need for replication. the expression of the eukaryotic translation initiation factor h (eif- h) (b- ) is up-regulated by infectious and inactivated vacv ihd-w. the protein eif- h stimulates protein biosynthesis, atp hydrolysis and helicase activity of eif- a. enzymatic investigations show that the affinity of eif a to rna is increased two-fold and helicase activity four-fold by an interaction with [ ] . the regulation of eif- h may contribute to an enhancement in protein biosynthesis which is required by the virus' need for protein expression. further on an increased expression of nuclear protein hcc- (cip ) (b- ) is denoted in both types of infected hek cells. this protein possesses a binding domain for ss and dsdna and is postulated to be part of the ribonuclein complex. interactions with the rna-helicases ddx and bat are verified which proves the influence of hcc- on the transcription of dna. the overexpression of hcc- in hek cells is known to decrease the cells' growth rate [ , ] . several members of the spliceosome, heterogeneous nuclear ribonucleoprotein b (b- ), novel protein similar to small nuclear ribonucleo-protein polypeptide a (b- ), heterogeneous nuclear ribonucleoprotein m (b- ), are modulated in both types of vacv ihd-w-infected hek cells. this heterogeneous protein complex splices introns of the hnrna. since the poxvirus genome has no introns and splicing of mrna is therefore obsolete, viral effectors may control the cells' protein biosynthesis by interfering with the cellular hnrna processing [ , ] . far upstream element-binding protein (fubp ) (b- ) stimulates the expression of transcription factor c-myc by binding onto far upstream element (fuse) upstream of the c-myc promoter [ ] . the protein is a known link between the apoptosis cascade and the c-myc oncogene, it further possesses helicase activity for dsdna. experiments with transfected cells show that a high fubp expression increases the expression level of c-myc and in this way protects the cell from apoptosis, while caspase-mediated cleavage of fubp induces apoptosis [ ] . both infectious and inactivated vacv ihd-w enhance the expression of fubp . ewing sarcoma breakpoint region (b- ) is a multifunctional protein with essential functions in gene expression, signal transduction, and mrna transport and processing. mutations in the protein-expressing gene lead to the development of ewing sarcomas and other tumours. the protein expression is suppressed after an infection with uv-inactivated as well as with active viruses [ ] . prefoldin subunit (b- ) is part of a heterohexamer chaperon (prefoldin) which binds cytosolic chaperonin (c.cpn) and transports target proteins to the hexamer. it is also involved in the folding of nascent peptides. gene deletions of prefoldin result in a dysfunction of the actin-tubulin cytoskeleton [ ] . prefoldin subunit is exclusively identified in cells infected with uv-inactivated vacv. the described alterations caused by the infection are results of the fact that the virus is taking control of the cells' gene expression as well as the protein biosynthesis and so aims for effective expression of viral proteins and the control of the cellular response. several viruses in general and poxviruses in particular are known to modulate the hosts' apoptosis pathways in order to avoid the antiviral defence mechanism at a cellular level [ ] . this results in an altered expression profile of apoptosis-relevant genes caused by viral gene products. during vacv transcription double-stranded rna (dsrna) is synthesised, since the virus possesses overlapping genes on both strands of its dna. the presence of dsrna activates the dsrna-dependent serine/ threonine kinase (pkr) which phosphorylates the α-subunit of the translation initiation factor eif- which is essential for protein biosynthesis. this phosphorylation inhibits the translation of mrna and thereby induces apoptosis [ ] [ ] [ ] [ ] . an altered modification on the γ-subunit of the translation initiation factor eif- (b- , b- ) caused by the infection was detected by a spot migration in the -de gel the relevance of which has not yet been determined in the literature. the dsrna-dependent pkr is usually inhibited by nucleophosmin (c- ) which is ubiquitously expressed in human cells and translocates between nucleus and cytoplasm [ ] . the downregulation of nucleophosmin (c- ), which can not be detected in cells infected with active vacv ihd-w, suggests that it is a cellular response to the infection for the purpose of anti-viral defence. this immune response is undercut by the product of the vacv e l gene, the putative double-stranded rna binding protein (d- ) which binds dsrna and inhibits apoptosis induction by preventing pkr from phosporylating the γ-subunit of the translation initiation factor eif- [ , ] . the e l gene product has been identified in hek cells infected with active vacv ihd-w, which is in correlation to proteome analysis of vacv virions that indicate that the putative double-stranded rna binding protein (d- ) is not present in the virion but is expressed in the early replication phase. the present proteome analysis has led to the identification of several further apoptosis-relevant proteins. the expression of two isoforms of prohibitin (c- , c- ) is decreased after infection with active vacv ihd-w. prohibitin regulates cell division, inhibits dna synthesis and sensibilises cells for apoptosis by destabilising the mitochondrial membrane [ ] . beyond that, prohibitin enhances the transcription of p [ ] . the infection of hek cells with active and inactivated vacv ihd-w inhibits the expression of the protein guanine nucleotide-binding protein subunit beta -like (c- ) which is an acceptor for activated protein kinase c (pkc). pkc is then bound to the cytoskeleton and conveyed to its target proteins, the marcks proteins. the receptor is also engaged in the regulation of the src kinase which can inhibit apoptosis by a phosphorylation of caspase- [ , ] . the reduced expression of the proteasome subunit alpha type- -like (c- ) can be seen as a further cellular defence strategy. the proteasome subunit alpha type- like (c- ) is part of the proteasome complex which cleaves peptides proteolytically at the amino acids arg, phe, tyr, leu and glu [ ] . the inhibition of the proteasome in tumour cell lines induces apoptosis. the phosphatidylethanolamine-binding protein (c- , c- ) is modified by an infection with active and inactivated vacv ihd-w, which results in an altered pi value. the protein binds to phosphatidylethanolamine which is presented at the surface of apoptotic cells, inhibits the rafkinase, prevents the activation of the map-kinase-cascade and the antiapoptotic transcription factor nf-b [ , ] . it is to be postulated that the protein modification is associated with an altered physiological activity. in this comparative proteome analysis the kda cell fusion protein (d- ) is identified in hek cells infected with active vacv ihd-w. its presence is predicted according to swissprot [ ] . the variola homologue of the a l gene is known to play an import role in virus penetration by fusing the outermost of the golgi-derived membranes enveloping the virus with the cells' plasma membrane. as expected, the major core protein p a (d- ) of vaccinia virions is further identified in both infected cell culture approaches [ ] . peroxiredoxin- (e- ) is an antioxidative enzyme which reduces hydrogen-and alkyl-peroxides and supports the antiviral activity of cd (+) t-cells [ ] . in contrast to the negative control, protein expression is enhanced by an infection with active and uv-inactivated viruses. transgelin- (sm -alpha homologue) (e- ) has not been functionally characterised yet. the protein possesses an actin-binding domain and is overexpressed in cells infected with both infectious and inactivated vacv ihd-w. it seems as if human transgelin is packed into vaccinia virions and transferred from cell to cell for an unknown reason [ ] . it can be hypothesised that it may serve as an anchor for the virus to move along the cytoskeleton. sorting nexin (e- ) has a binding domain (px domain) for phospholipids and transports proteins within cells from organelles to membranes [ ] . the protein expression is enhanced after infection of hek cells with active and uv-inactivated vacv ihd-w. profilin (e- ) binds actin and participates in the build-up of the cytoskeleton. as a response of extracellular signals, high concentrations of profilin inhibit the actin-polymerization which is enhanced by low profilin concentrations [ ] . both types of infection increase expression of human profilin, while vacv possesses a profilin homologue by itself. chemical assisted fragmentation (caf) of peptides by nterminally sulfonation with spitc improves confidence in protein identification from psd spectra. ziptipμ-c columns solid phase-supported spitc derivatization is preferable compared to an in-solution approach, since s/n ratios as well as signal intensities are increased, and so the sensitivity for recording high quality psd spectra is enhanced. proteome analysis of vacv ihd-w-infected hek cells in the late replication phase shows the upregulation of the energy metabolism and alteration of gene expression regulation. further on the modulation of apoptosis which counteracts the cellular anti-viral response is demonstrated by the regulation of several proteins from different signalling pathways. interestingly the effects of uv-inactivated viruses and infectious viruses on the hosts' proteome are quite similar. there is no difference between the identified alterations of the energy metabolism and just one differentially expressed protein associated with gene expression and protein biosynthesis (b- ). therefore the up-regulation of the energy metabolism and alterations in the regulation of the cellular gene expression are results of virus entry and virions' proteins. however, the modulation of apoptosis is clearly affected by effects that correlate with infectious viruses. the absence of nucleophosmin in cells infected with infectious viruses, for example, may be the response on viral dsrna resulting from transcription of viral dna, as the competing apoptosis modulator putative double-stranded rna binding protein (d- ) is not a part of the virion. in part, the identified proteins have not yet been described in the context of poxvirus infections and need to be further characterised using virological methods to identify their meaning for apoptosis modulation and pathogenesis. all of the electrophoresis apparatus (ipgphor ii, multiphor ii chamber electrophorese, eps power supply) were purchased from amersham pharmacia biotech (uppsala, sweden). ipg drystrips and pharmalyte ph - virus stock solution containing . × pfu was centrifuged for h at , × g and °c. the virus pellet was resuspended in ml phosphate buffered saline (pbs), transferred into a -well plate and inactivated by uv irradiation of . w/m in a stratalinker . success of inactivation was checked by immunofluorescence staining of infected cells. the two-dimensional polyacrylamide gel electrophoresis with immobilised ph gradient was performed according to the procedure by görg et al [ ] . initially, μg dried protein was solubilised in μl rehydration solution in the second dimension the proteins were separated in a -%-polyacrylamide gel ( × × mm ) at w, v and ma for h at °c in a horizontal system. the size marker was rotimark ® - kda (roth, karlsruhe, germany) and a tris-glycine buffered system according to laemmli ( ) was used [ ] . after electrophoresis, the proteins were silver stained with the proteo-silver plus silver stain kit (sigma-aldrich) according to the manufacturer's instructions. differences in the protein expression profile of the -d gels were identified by use of the image analysis software melanie . (swiss institute for bioinformatics). a quantitative analysis was disclaimed since silver staining is not an end point method. a match set was created from scanned gel images in order to compare protein spots across gels. the gel containing the best resolution and most spots was chosen as the master gel. landmark spots were defined and manual editing of the matching procedure was performed to improve the automated matching results. finally, the mismatched spots representing differences in protein expression were manually checked and selected for mass spectrometric analysis. differences in the protein expression profile of the -d gels were identified by use of the image analysis software melanie . (swiss institute for bioinformatics, lausanne, switzerland, http://www.expasy.org/melanie). a quantitative analysis was disclaimed since silver staining is not an end-point method. spots representing differences in protein expression were excised as dice with mm edge length each, which were destained with the proteosilver™ plus silver stain kit (sigma-aldrich) according to the manufacturer's instructions. gel dice were then dried in a speedvac after dehydration in μl acn. the dried dice were swollen in μl reaction buffer ( mm ammonium bicarbonate [nh hco ] ph . ) containing ng trypsin (trypsin profile igd kit, sigma-aldrich) for min at °c, before another μl reaction buffer was added. the tryptic digest was incubated overnight at °c. the proteolytic peptide containing supernatant was transferred into a new reaction tube. peptides remaining in the gel dice were extracted by adding μl : acn/ . %tfa for min. pooled peptides were evaporated to dryness in a speedvac for further analysis. reduction of disulfide bonds was done by addition of μl m dithiotreitol (dtt) to μl . μg/μl bsa in mm nh hco ph . at °c for min. subsequently free thiol groups were carboxyamidomethylated by adding μl m iaa at °c for min in the dark. excessive iodoacetamide (iaa) was converted with μl m dtt at °c for min before the proteolytic digest was induced by addition of μg trypsin ( : w/w) and incubated for h at °c. for spitc derivatization the desired amount of protein was transferred to a microcentrifuge tube and evaporated to dryness in a speedvac. n-terminal peptide derivatization with -sulfophenyl isothiocyanate on ziptipμ-c columns dried peptides were resolved in μl . % tfa, . μl of which were mixed with . μl matrix ( mg α-cyano- hydroxycinnamicacid [chca] in ml acn/ . % tfa [ : v/v]) on a stainless steel carrier and were air-dried. prior to the recording of peptide mass fingerprints, the spots were washed with . μl mm biammonium citrate three times. the remaining peptide solution was bound to a ziptipμ-c (millipore) pipette tip which was wetted with μl acn/ . % tfa ( : v/v) and washed twice with μl . % tfa. subsequently the ziptipμ-c was loaded with μl mg/ml -sulfophenyl isothiocyanate (spitc) in mm nahco ph . and incubated at °c for h in a circulating air oven. derivatised peptides were washed five times with . % tfa and three times with mm biammonium citrate prior to elution with μl acn/ . % tfa ( : v/v) directly onto the maldi target. . μl matrix ( mg chca in ml acn/ . % tfa [ : v/v]) was added and the spot was air-dried. dried peptides were resolved in μl derivatization solution ( mg/ml -sulfophenyl isothiocyanate [spitc] in mm nahco ph . ) and incubated at °c for min. purification, elution and co-crystallization of derivatised peptides was done by use of ziptipμ-c (millipore) pipette tips as described above. the mass spectrometric investigations have been carried out using an axima-cfr plus (kratos analytical, manchester, uk). peptide mass fingerprints were recorded in positive ion reflectron mode with a setting of the laser intensity of . μj and a maximum laser-repetition rate of . hz at a pulse extraction of m/z (delay-time). each recorded spectrum was the sum range of profiles, accumulated from five laser shots. ten m/z values greater than were automatically selected with descending intensity by the launchpad software (kratos analytical) for post source decay (psd) measurement. profiles accumulated from ten laser shots were accumulated for one psd spectrum which was recorded in positive ion reflectron mode with a laser intensity of . μj and a maximal laser-repetition rate of . hz at a pulse extraction of m/z (delay-time). an external -points calibration was performed by using appropriate peptide standard (calibration mix [proteomix] - da [laserbio labs]) prior to each measurement. a combined data file of m/z values of the pmf and psd spectra was sent to the expasy server (swiss institute for bioinformatics) and synchronised with the swissprot database for protein identification. peaks from the raw data were picked using mascotdistiller (matrix science, boston, usa) with a minimal signal/noise of and a peak width of . m/z values were annotated with a mass tolerance of +/- . da in ms and +/- . da in psd mode. carbamidomethylated cystein residues and spitc derivatised n-termini were set as fixed modifications, oxidised methionine residues as a variable modification. the challenge of emerging and reemerging infectious diseases rapid response research to emerging infectious diseases: lessons from sars human monkeypox: an emerging zoonosis cowpox virus infection: an emerging health threat potential biological weapons threats dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the a r gene a l protein mediates vaccinia virus interaction with cell surface heparan sulfate cell surface proteoglycans are necessary for a l protein-mediated cell fusion: identification of the n-terminal bartel et al region of a l protein as the glycosaminoglycan-binding domain vaccinia virus envelope d l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells vaccinia virus envelope h l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo pox proteomics: mass spectrometry analysis and identification of vaccinia virion proteins analysis of vaccinia virushost protein-protein interactions: validations of yeast two-hybrid screenings de novo sequencing of tryptic peptides sulfonated by -sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry improved protein identification efficiency by mass spectrometry using n-terminal chemical derivatization of peptides from angiostrongylus costaricensis, a nematode with unknown genome determination of aldolase in animal tissues the isoenzymes of malate dehydrogenase and their regulation in saccharomyces cerevisiae ecto-f fo atp synthase/ f atpase: metabolic and immunological functions mutations and polymorphisms of the gene encoding the beta-subunit of the electron transfer flavoprotein in three patients with glutaric acidemia type ii further: biochemical and kinetic characterization of human eukaryotic initiation factor h hcc- is a novel component of the nuclear matrix with growth inhibitory function growth inhibitory effect of hcc- /cip is associated with induction of apoptosis, not just with g /m arrest structure and expression of the gene (hnrpa b ) encoding the human hnrnp protein a /b purification and characterization of native spliceosomes suitable for three-dimensional structural analysis a sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new dna-binding motif far upstream element-binding protein- , a novel caspase substrate, acts as a cross-talker between apoptosis and the c-myc oncogene ewing sarcoma fusion protein ewsr / fli interacts with ewsr leading to mitotic defects in zebrafish embryos and human cell lines prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin rice ap: interferon mediated, double-stranded rna-dependent proteine kinase is inhibited in extracts from vaccinia virus-infected cells the dsrna proteine kinase pkr: virus and cell control induction of apoptosis by the dsrna-dependent protein kinase (pkr): mechanism of action characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon induced proteine kinase nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor kinase pkr the e l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase vaccinia virus (strain ioc) putative double-stranded rna binding protein prohibitins control cell proliferation and apoptosis by regulating opa -dependent cristae morphogenesis in mitochondria prohibitin induces the transcriptional activity of p and is exported from the nucleus upon apoptotic signaling essential role of pkc[delta] in apoptosis induction of mouse thymocytes src kinase phosphorylates caspase- on tyr : a novel mechanism of apoptosis suppression the mgc project team: the status, quality, and expansion of the nih fulllength cdna project: the mammalian gene collection (mgc) exposure of phosphatidylethanolamine on the surface of apoptotic cells raf- kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis uniprotkb/swiss-prot p (vfus_vaccc) integrated into uniprotkb/ swiss-prot the complete dna sequence of vaccinia virus plasma and red blood cell protein maps: update comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions snx regulates endosomal function through its px-domain-mediated interaction with ptdins( )p distinct biochemical characteristics of the two human profilin isoforms the current state of two-dimensional electrophoresis with immobilized ph gradients cleavage of structural proteins during the assembly of the head of bacteriophage t proteome analysis of vaccinia virus ihd-w-infected hek cells with -dimensional gel electrophoresis and maldi-psd-tof ms of on solid phase support n-terminally sulfonated peptides author details friedrich-alexander university erlangen-nuremberg, institute of bioprocess engineering, henkestraße , erlangen, germany. robert koch-institute, center for biological security , nordufer , berlin, germany.authors' contributions jd carried out the two-dimensional gel electrophoresis, image analysis as well as the mass spectrometric measurements. sb established the method for fragmentation of spitc derivatized peptides and executed the database searches for protein identification. the manuscript was drafted by sb and jd in equal parts. the cell culture part of this study was performed by db, whereas kd supported the image analysis and database searches. rb financed the analytical part of the study. hl coordinated the study and gave relevant contributions to the technical background. an conceived the study, and participated in its design. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -u zpyy authors: tan, bing; wu, li-jun; yang, xing-lou; li, bei; zhang, wei; lei, yong-song; li, yong; yang, guo-xiang; chen, jing; chen, guang; wang, han-zhong; shi, zheng-li title: isolation and characterization of adenoviruses infecting endangered golden snub-nosed monkeys (rhinopithecus roxellana) date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: u zpyy background: adenoviruses are important pathogens with the potential for interspecies transmission between humans and non-human primates. although many adenoviruses have been identified in monkeys, the knowledge of these viruses from the colobinae members is quite limited. findings: we conducted a surveillance of viral infection in endangered golden snub-nosed monkeys (rhinopithecus roxellana) in the subfamily colobinae in china, and found that . % of sampled individuals were positive for adenovirus. one of the adenoviruses (sadv-wiv ) was successfully isolated and its full-length genome was sequenced. the full-length genome of wiv is , bp in size, has a g + c content of . %, and encodes putative genes. sequence analysis revealed that this virus represents a novel species in the genus mastadenovirus. diverse cell lines, including those of human origin, were susceptible to wiv . conclusion: we report the first time the isolation and full-length genomic characterization of an adenovirus from the subfamily colobinae. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. within the family adenoviridae, the genus mastadenovirus contains a group of non-enveloped icosahedral viruses that range in size from to nm and contain a linear double-stranded dna genome of approximate kb [ ] . members of this genus are pathogens that infect a wide range of mammals. in humans, adenoviruses (advs) cause a variety of pathologies including acute respiratory illness, epidemic keratoconjunctivitis, acute haemorrhagic cystitis, hepatitis, myocarditis, and gastroenteritis [ ] . although usually self-limiting, adv infection may induce serious morbidity and mortality, especially in immunocompromised patients and transplant recipients [ ] . similarly, advs have been associated with diarrhea, acute respiratory illness, pneumonia, and hepatitis in captive non-human primates [ ] [ ] [ ] . in , an outbreak of adv-infection led to % fatality in titi monkeys of the genus callicebus, a group of new world monkeys [ ] . notably, neutralizing antibodies against this titi monkey mastadenovirus were detected in two human individuals, indicating the potential for zoonotic transmission. several other cases of potential adv transmission between humans and non-human primates have also been documented recently [ ] [ ] [ ] . to date, at least distinct types of simian adenoviruses (sadvs) have been reported. most of them infect captive great apes and members of the subfamily cercopithecinae [ , ] . sadvs that infect great apes are closely related to types that infect humans, which belong to the species human mastadenovirus a to human mastadenovirus f (hadv-a-f) (primarily b, c, and e) [ , ] ; those infecting the cercopithecinae members have been classified into hadv-g, species simian mastadenovirus a (sadv-a), and the recently proposed species sadv-b-h [ , , ] . it should be noted that the group of old world monkeys (owms) comprises two subfamilies, the cercopithecinae and the colobinae; however, knowledge of advs from the latter sub-group remains quite limited. only a few short sequences of adv dna have been reported in this subfamily [ ] . golden snub-nosed monkeys (rhinopithecus roxellana) living in shennongjia nature reserve (snr) in hubei, china, are an endangered species belonging to the subfamily colobinae [ ] . visitors to snr have close contact with monkeys, which are fed by animal nurses in the reserve area; this raises the possibility of viral transmission between humans and monkeys. however, despite the extensive efforts to protect these monkeys from being endangered, viruses infecting these animals are poorly studied. in this study, we conducted a surveillance of viral infections in faecal samples from r. roxellana collected from snr in . pan-pcr analysis was performed to detect the presence of advs, coronaviruses, enteroviruses, mammalian reoviruses, and rhinoviruses [ ] [ ] [ ] . except the samples that tested positive for adv, all samples were negative for the tested viruses. the virus strain in the positive samples shared % nucleotide identity, based on the -bp dna polymerase gene sequences. one of them was successfully isolated and cultured in vero e cells (fig. ) and its full-length genome was sequenced. following the order of viruses isolated in our laboratory at wuhan institute of virology (wiv), we tentatively named this isolate as simian adenovirus wiv (sadv wiv ). the wiv genome comprises , bp, including . % a, . % c, . % g, and . % t (genbank accession number kx ) (fig. ) . its -bp extreme ends are inversely repeated, and start with the conserved motif catcatcaat [ ] . the genome was predicted to encode proteins and a virus-associated rna (fig. ) . although some primate advs are known to encode two fibre genes, only one copy was identified in the genome of wiv . similarly, a single gene of noncoding virus-associated rna was identified between the second exon of ptp and the k gene. except for the absence of the e . k gene, organization of the wiv genome was identical to that of sadv-a and g isolates infecting owms. the putative gene products of the wiv genome displayed - % amino acid (aa) identities to their closest homologues encoded by members of sadv-a (additional file : table s ). most genes displayed > % aa identity with those encoding structural proteins, including piiia, penton base, pvii, pvi, pviii, and hexon. by contrast, fibre protein showed only % aa identity to its closest homologue, which may suggest distinct infectivity in vivo due to its critical role in host cell binding [ ] . dna [ ] . c. mona, cercopithecus mona; c. aethiops, chlorocebus aethiops; c. guereza, colobus guereza; p. cynocephalus, papio cynocephalus; p. h. anubis, papio hamadryas anubis; p. badius, piliocolobus badius. the phylogenetic trees were constructed using the program mega, version . , using the neighbor-joining method with , bootstrap replicates. percentage bootstrap values > are indicated at the nodes. scale bars indicate evolutionary distance polymerase is usually used for classification of adv [ ] . based on analysis of sequences of this gene, wiv displayed the highest aa identity ( %) to sadv- , suggesting that it is a distinct species. comparison of protein sequence similarities suggested that wiv is distantly related to owm advs. phylogenetic trees were constructed for better evaluation of the evolutionary relationship between wiv and the reported advs. consistently, wiv formed a separate branch closer to a common ancestor of sadv-a and g based on analyses of both dna polymerase and penton base sequences (fig. a, b) . when we used the partial hexon sequences available in all owm types, a common ancestor of three colobinae types (jn , jn , and jn ) was identified as the nearest neighbor to wiv (fig. c) . these observations suggested coevolution between wiv and golden snub-nosed monkeys. homologous recombination has been demonstrated to be an important evolutionary mechanism for advs, especially for hadv-d members [ ] . however, no signals of recombination were identified in the wiv genome, using rpd and simplot software [ , ] . for investigating the antibody prevalence against the adv infection, virus neutralization assay was performed using archived serum samples collected from golden snub-nosed monkeys in snr [ ] . as a result, crossneutralizing antibodies against wiv were identified in all the samples at titers ranging from to . the potential host range of wiv was evaluated using cell infectivity assays. seven cell lines were tested: a (human lung, atcc crm-ccl- ), hek- t (human embryonic kidney, atcc crl- ), nci-h (human lung, atcc crl- ), llc-mk (macaca mulatta kidney, atcc ccl- ), msin (miniopterus schreibersi intestine), rlki (rousettus leschenaulti kidney), and rski (rhinolophus sinicus kidney) [ ] . cytopathic effects were observed in llc-mk , rski, and all three human cell lines, indicating that wiv has a wide host range. in conclusion, we described here the first isolation and characterization of a replication-competent adv from monkeys in the subfamily colobinae. the cumulative prevalence of adv in endangered golden snub-nosed monkeys from snr was found to be . %. the present isolate from these monkeys is distantly related to known adv types, and likely represents a novel species in the genus mastadenovirus. its ability to infect diverse cell lines, including those of human origin, highlights the potential risk of new and emerging infections in tourists visiting snr. result from the virus neutralization assay suggested that the infections of wiv may be highly prevalent in these monkeys. further studies are required to determine its host range and role in causing disease in both golden snub-nosed monkeys and humans. long-term surveillance of viral prevalence in this region should be conducted in the future. additional file : table s . predicted genes of wiv and comparison to those of known adenoviruses. (docx kb) advs: adenoviruses; hadv: human mastadenovirus; owms: old world monkeys; sadv: simian adenovirus or mastadenovirus; snr: shennongjia nature reserve; wiv: wuhan institute of virology virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses human adenovirus: viral pathogen with increasing importance adenoviruses in immunocompromised hosts a novel adenovirus species associated with an acute respiratory outbreak in a baboon colony and evidence of coincident human infection detection of viral agents in fecal specimens of monkeys with diarrhea cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony new adenovirus species found in a patient presenting with gastroenteritis multiple cross-species transmission events of human adenoviruses (hadv) during hominine evolution isolation and characterization of adenoviruses persistently shed from the gastrointestinal tract of non-human primates adenovirus and herpesvirus diversity in free-ranging great apes in the sangha region of the republic of congo novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions taxonomy proposal for old world monkey adenoviruses: characterisation of several non-human, non-ape primate adenovirus lineages genome analysis of four old world monkey adenoviruses supports the proposed species classification of primate adenoviruses and reveals signs of possible homologous recombination rhinopithecus roxellana, the iucn red list of threatened species host range, prevalence, and genetic diversity of adenoviruses in bats simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays isolation and identification of bat viruses closely related to human, porcine, and mink orthoreoviruses genomic analyses of recombinant adenovirus type a in china structural studies on adenoviruses homologous recombination in e genes of human adenovirus species d full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination rdp : recombination detection and analysis from sequence alignments isolation and characterization of a bat sars-like coronavirus that uses the ace receptor novel bat adenoviruses with an extremely large e gene molecular detection of novel adenoviruses in fecal specimens of captive monkeys with diarrhea in china this work was jointly funded by the national natural science foundation of china the assembled sequence data were uploaded to the genbank database (http://www.ncbi.nlm.nih.gov/nuccore) at the accession number of kx . authors' contributions bt, ljw, ysl, yl, gxy, jc, gc, hzw, and zls collected samples. xly, bl, and wz performed the pcr assays. bt performed the other experiments and analyzed the data. bt and zls conceived of the study and drafted the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - qdnnwvd authors: zhang, xuefeng; wang, jing; lu, jing; li, rongrong; zhao, shuli title: immunogenicity of adenovirus-vector vaccine targeting hepatitis b virus: non-clinical safety assessment in non-human primates date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: qdnnwvd background: a new promising therapeutic approach has emerged for patients chronically infected by the hepatitis b virus (hbv) with the development of a non-replicative adenovirus vector vaccine candidate (ad-hbv). the vaccine encodes a fusion protein composed of a truncated hbv core protein, mutated polymerase protein, and two envelope domains. in this study, we assessed the immunogenicity of ad-hbv administered to cynomolgus monkeys during a non-clinical safety assessment. methods: the virus was subcutaneously administered at . × ( ) viral particles (vp)/animal (low-dose group), . × ( ) vp/animal (mid-dose group), and . × ( ) vp/animal (high-dose group); the control groups were administered an ad -null virus ( . × ( ) vp/animal) and saline only. results: except for inflammatory cell infiltration under the skin at the injection sites and transient elevation of body temperature and serum albumin, no ad-hbv-related toxic effects were noted in any treatment group. moreover, interferon (ifn)-γ enzyme-linked immunospot assays showed that ad-hbv induced the targeting of t cells to a broad spectrum of hbv-specific epitopes spanning all three of the selected hbv immunogens (core, polymerase, and envelope domains) in a dose-dependent manner. although anti-ad antibody was produced in all groups (except for the saline control), the antibody titers were significantly lower in the high-dose ad-hbv group than in the group that received the same dose of the ad-null empty vector. in addition, the ifn-γ and il- expression levels in the liver were significantly improved for the mid-dose, high-dose, and ad-null control group (p < . ), but not for the low-dose group. conclusions: taken together, this safety assessment indicates that the ad-hbv candidate vaccine is a potent specific immunotherapeutic agent, supporting its further clinical development as an anti-hbv infection vaccine. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. hepatitis b virus (hbv) infection continues to be a serious clinical challenge, which induces the development of liver cirrhosis and hepatocellular carcinoma, even leading to mortality, in many areas of the world [ , ] . currently, the utility of first-line antiviral agents or immune system modulators recommended by the world health organization can only help to reduce and control the viral load and minimize liver damage, but cannot effectively clear the infection and seldom achieve a cure [ , ] . thus, these current therapies remain far from satisfactory. in particular, the wide spread of multidrug-resistant hbv strains is increasingly threatening the efficacy of currently available antiviral drugs [ , ] . therefore, there is an urgent need to actively develop and test promising novel therapies to target hbv. gene therapy is considered to be a newly effective option against many diseases by directly delivering therapeutic genes or those that can induce an improvement in the systemic immune response [ ] . owing to its efficient infectivity, high loading capacity, and stability outside of cells, adenovirus (ad) is a promising candidate vector for gene delivery, and has been widely adopted for many gene therapy strategies developed to date [ ] . ad-hbv is considered to be the primary hbv immunotherapeutic developed through a transgenetic approach with the aim of application for treating patients chronically infected by hbv [ , ] . ad-hbv is a therapeutic vaccine candidate based on human e -and e -deleted replication-defective ad serotype (ad ) vectors encoding an hbv fusion protein consisting of a truncated form of the hbv core domain (at the n-terminal) fused to a deleted and mutated hbv polymerase sequence, and two hbv envelope domains are inserted within the polymerase deletions. the nucleotide sequence encoding this hbv fusion protein is based on the hbv isolate y , which was slightly modified [ ] . this vaccine candidate was designed to stimulate specific t cell-based immunity similar to that seen in hbv resolvers. pharmacodynamics studies on ad-hbv have been performed in three animal models thus far: hla-a transgenic mice, c bl mice, and balb/c mice. in all cases, ad-hbv was demonstrated to effectively induce t cells to produce various cytokines [interferon (ifn)-γ/ tumor necrosis factor (tnf)-α and interleukin (il)- ] in response to hbv antigen stimulation [ ] . however, there were some potential disadvantages noted, including damage to the host immune response, hepatocytoxicity, and a short half-life; thus, non-clinical safety assessment is essential prior to further clinical development and trial as a vaccine. an unsolved challenge in the development of gene therapy drugs is that the vectors and their encoding proteins ultimately become immunogenic in some patients, thereby provoking an immune response resulting in immunotoxicity [ ] . in contrast to therapeutic proteins whose immunogenicity is mainly evaluated by measuring the levels of anti-drug antibodies produced, there is currently no well-established and widely accepted method for the immunogenicity assessment of gene therapy drugs [ , ] . therefore, in the present study, we focused on evaluating the immunogenicity of the subcutaneous injection of ads-hbv vaccines in a non-human primate at different doses to obtain reference data through a non-clinical safety assessment in preparation for a clinical trial. the human lung cancer cell line a was purchased from shanghai cell resource center of the chinese academy and cultured in dulbecco's modified eagle medium (dmem)/f (thermo fisher biochemical products, beijing, china) supplemented with antibiotics ( u/ml penicillin and . mg/ml streptomycin; invitrogen, life technologies) and % fetal bovine serum (gibco, life technologies) at °c in a % co incubator. all ad vectors used in this study, including ad not encoding any antigen (empty ad, hereafter ad-null), ad -luciferase (ad -luc ), and ad-hbv, were constructed, produced, purified, and titrated as described elsewhere [ ] [ ] [ ] . finally, the purified viruses were re-suspended in sterile phosphate-buffered saline (pbs) buffer and stored at − °c. virus quantification (number of virus particles, vp) was determined by high-pressure liquid chromatography. the long-term toxicity of ad-hbv vaccines was evaluated in b virus-and ad-free cynomolgus monkeys. macaca fascicularis as the non-human primate model for the present safety assessment, which were obtained from hainan new source biotech co., ltd. (hainan, china). a total of monkeys were used in this study, with equal numbers of males and females, weighing . - kg ( . - -year-old). in brief, the monkeys were divided into five groups (n = per group, five animals per sex) and injected with ad-hbv at the following doses: . × vp/animal (low-dose group), . × vp/animal (mid-dose group), and . × vp/animal (high-dose group). vehicle control ( ml of . % saline per animal) and injection of . × vp/animal ad -null were used as controls. all animals were injected subcutaneously weekly seven times in total, and the animals' clinical symptoms were continually observed twice a day for months. after gene therapy ( days after the last dosing), six animals from each group (three animals per sex) were sacrificed for safety, toxicology, biodistribution, and other analyses. the remaining animals were left to recover for weeks. monitoring parameters were collected from each animal at several time points. the time schedule and experimental protocol are summarized in table . to evaluate the immunoreaction of ad-hbv, the lymphocyte subgroups in the peripheral blood were analyzed throughout the trial by a flow cytometry assay [ ] . peripheral blood lymphocytes from all animals were isolated using lymphocyte separation medium (tbdscience, tianjin, china) density-gradient centrifugation according to the manufacturer's instructions. the cells were then stained with specific conjugated antibodies (anti-cd -pe, anti-cd -apc, anti-cd -pe cy , and anti-cd -apc) and subjected to flow cytometry, respectively. the data were analyzed using flowjo software. ihc was used to determine the expression of ifn-γ, tnf-α, and il- in the liver tissues collected from animals in each group. in brief, paraffin-embedded tissues were prepared from sacrificed animals, and -μm sections of liver tissues were prepared by extracting the paraffin in xylene. the sections were then re-hydrated, the endogenous peroxidase was inactivated by % h o pbs, and then sections were treated with antigen repair solution ( mm citrate solution; maixin-bio, fujian, china) in a microwave oven at - °c for min to retrieve the antigens. after blocking non-specific reactivity by % bovine serum albumin, the sections were incubated with antibodies against monkey ifn-γ (ab , abcam), tnf-α (ab , abcam), and il- ( , , biolegend) for h at °c. after washing three times with pbs, the suitable secondary antibody labeled with horseradish peroxidase (hrp) was added to the sections and incubated for h at °c. the slides were then washed five times with pbs, and developed using a diaminobenzidine kit (maixin-bio, fujian, china) according to the manufacturer's instructions. immunoreactivity was graded into the following four groups based on the frequency of positive staining: − (negative), no specific staining or < % positive staining; +, ≥ to < % positive staining; ++, ≥ to < % positive staining; and +++, ≥ % positive staining. grades − and + were considered to reflect low expression, and grades ++ and +++ were considered to reflect high expression [ ] . the quantity of ifn-γ-secreting t cells in the peripheral blood during this trial was assessed by elispot using the monkey ifn-γ kit ( m- hpw- , mabtech inc., cincinnati, oh, usa) following the manufacturer's instructions [ , ] . the peptides used in this study ( table ) were previously identified as showing reactivity in a pilot experiment with cynomolgus monkeys, and were synthesized by genscript biological technology co., ltd. (nanjing, china). for each library, peptides were dissolved in % dimethyl sulfoxide (dmso, sigma) and pooled at a concentration of mg/ml per peptide, and each pool was tested at a final concentration of μg/ml per peptide in the elispot assays. three peptide pools were used as stimulatory agents. hence, the elispot assays were carried out in five groups, including the negative control (unrelated peptide in dmso, dlmdlmgyiplv), core pool, env pool, pol pool, and positive control (anti-cd igg, : , provided in the kit). in brief, approximately ml of peripheral blood was collected to isolate the lymphocytes from each sample in tubes with heparin. lymphocytes ( × /well) in triplicates were plated and incubated with complete dmem containing different peptides or control agents for h in a °c humidified incubator with % co . the medium was then removed and the plates were washed five times with pbs. hrp-conjugated anti-ifnγ ( μl, : ) was added to each well, and the plates were washed five times with pbs after incubation for h at room temperature. finally, tmb substrate solution was used to detect the ifnγ-positive spots. after the plate dried, the spots in each well were inspected and counted under a dissection microscope. the anti-ad-hbv antibody titers were measured by indirect enzyme-linked immunosorbent assay (ielisa) as previously described [ ] . in brief, binding of the anti-drug antibodies in serum samples with pre-coated inactivated antigen on a microtiter plate ( . × vp/ μl carbonate/bicarbonate buffer per well) was detected with hrp-labeled anti-monkey immunoglobulin g (igg). based on a cut-off point of optical density values at nm, the positive and negative pre-administration serum samples from the same animals were screened out and their neutralizing activity was further measured using an adapted luciferase-based virus neutralization assay [ ] . in brief, the diluted serum samples ( / , / , and / ) were mixed with . × vp of ad -luc in complete dmem, which is a luciferase-expressing ad vector (shenzhao biotechnology, china), and incubated for h at room temperature. then, × a cells were added to the medium and incubated at °c, % co for h. the luciferase assay system (promega) was used to measure luciferase activity in the cells with a microplate reader (biotek). finally, neutralizing antibody activity in the blood of each animal after administration was evaluated according to the following formula. neutralization efficiency (%) = [(mean fluorescence intensity of the pre-administration control group − mean fluorescence intensity of the administration group)/mean fluorescence intensity of pre-administration control group] × %. data are presented as mean ± standard deviation of each group, which were analyzed using the spss . software packet. comparisons between groups were tested by one-way anova, measurement data was analyzed by the chi-square test, and rank data were analyzed by fisher's exact probability. data of dose groups were compared with those of the vehicle control group; p < . was deemed to reflect a statistically significant difference. erythema was observed at the injection site of the skin at different proportions in the animals of the ad-hbv groups (low-dose, / ; mid-dose, / ; high-dose, / ) and ad -null group ( / ), but was not detected in the vehicle control group. histological examination showed infiltration of inflammatory cells (plasma cells, lymphocytes) under the skin at the injection site, representing a local immune response caused by administration of the ad vector (fig. ) . compared with the vehicle control group, transient elevation of alb (day , , and ) and tp (day ) levels was detected along with an increase in body temperature (day , , , , , and ) in the ad-hbv and ad-null groups after injection. however, these levels did not change substantially before and after vector administration and fluctuated within the normal physiological range (additional file : table s , additional file : table s and additional file : table s ). thus, these effects did not reflect a significant toxicological reaction. compared to the vehicle control group, there was a significant difference in lymphocyte subsets detected for cd + t cells in the low-dose group and macrophages (cd + cd +) in the ad-null group; however, these changes were noted for a single index and were not dose-dependent, indicating that they were of minimal biological significance. the other parameters fluctuated within normal ranges throughout the study (additional file : table s ). in addition, throughout the course of the study, no significant differences were observed in body weight, overall, these findings demonstrated that the main toxicity of ad-hbv is related to stimulation at the injection site by the ad itself. to confirm the potent and functional cellular immune response in non-human primates resulting from ad-hbv injection during this non-clinical safety assessment, we monitored the hbv-specific immune response using ifn-γ elispot assays. although the numbers of hbv antigen (core, env, and pol)-specific ifnγ+ cells in the peripheral blood mononuclear cells (pbmcs) were significantly higher for all three dose groups (low, mid, and high) compared to those in the vehicle control group and ad -null control group (p < . ) at day , , and , the cell numbers in the low-dose group ( vp/animal) were significantly lower (p < . ) than those in the mid-dose ( vp/animal) and high-dose ( vp/ animal) groups (fig. ). in addition, although the the numbers of hbv antigen-specific ifnγ+ cells in pbmcs for the mid-dose group were significantly lower than those of the high-dose group at the first administration on day (p < . ), there was no difference between these groups at days and . these results indicated that the dose setting in this toxicity assessment test was reasonable, and vp/ animal was the highest effective dose in the ad-hbv test of cynomolgus macaques. the binding and neutralization activity of anti-drug antibodies to the ad vector-based vaccines may affect the clinical efficacy, alter the pharmacokinetic profiles, or cause adverse effects. therefore, we used the anti-ad antibody-negative animals in the non-clinical safety assessment test and monitored the levels of anti-ad antibody in the serum after ad-hbv administration. the levels of anti-ad antibodies dose-dependently increased after the administration of ad-hbv, and reached a peak value at day that was sustained until the last administration (day ). the levels of anti-ad antibodies in the ad -null control group (ad empty vector, vp/animal) were higher than those detected for the animals that received the same-dose (high-dose vp/ animal) of ad-hbv group, which may have resulted from gene inserting of truncated pol, core and env antigen. in addition, the antibody titers were sustained at higher levels after weeks of recovery (day ) (fig. ) . the neutralization activity of the highest-titers serum samples from every animal in the ad -null control group and the three ad-hbv groups (low, mid, and high doses) were then measured by the ad vector luciferase-expressing inhibition assay. at h of infection, the fluorescence intensity in the a cells gradient increased in proportion with the ad -luc concentration. to improve the sensitivity and sensitivity of this model, we selected vp/ml of ad -luc to infect a cells in the neutralization experiment. compared with the pre-dosing serum samples, diluted serum samples ( / , / , and / ) from all cynomolgus monkeys were injected subcutaneously with a low dose ( . × vp/animal), mid dose ( . × vp/animal), and high dose ( . × vp/animal) of ad-hbv, or with an empty ad-null vector ( . × vp/ animal); an equal volume of saline was injected as a negative control. hbv antigen-specific cells were monitored and quantified by interferon-γ (ifnγ) elispot assays conducted at day (a), (b), (c), and (d) post-injection using peripheral blood mononuclear cells (pbmcs) respectively stimulated by hbv peptide pools (core, env, pol , pol , and pol ) or irrelevant peptides (dlm). *p < . , **p < . vs. vehicle control anti-drug antibody-positive animals decreased the fluorescence intensity to different extents in the infected a cells by ad -luc . the serum neutralizing effects of the high-dose and ad-null groups were significantly higher than those of the low-dose and mid-dose groups, indicating that the anti-drug antibody-positive serum inhibited the infection activity of ad -luc and that the neutralization activity is dose-dependent (fig. ) . thus, although the anti-ad antibody was produced in almost all animals after the first immunization, these antibodies could partially neutralize the ability of ad infection. since ad vectors are predominantly sequestered by the liver after administration, and a previous study revealed that ad infection could significantly improve the expression of antiviral immune cytokines, we analyzed the levels of ifn-γ, il- , and tnf-α proteins in the liver by ihc to explore the immune response strategy in the liver. as shown in table and fig. , administration with ad-hbv significantly improved the ifn-γ and il- expression levels of the livers in the mid-dose, high-dose, and ad-null control group (p < . ), but not in the low-dose group. this suggested that the increased expression of these two cytokines in the liver should be related to the amount of ad vector injected. however, there was no correlation between the tnf-α expression and administration of ad-hbv. several studies have been conducted on the application of gene therapies using ad -based vaccines for the treatment of cancer or chronic virus infections, which have shown that the immunogenicity responses specific for ad proteins and for transgene-encoded proteins induced by vaccines could have a significant impact on the efficacy of a recombinant ad vaccine [ ] [ ] [ ] . thus, an essential aspect to ensure the efficacy of virus-vector drugs is to comprehensively and accurately evaluate their immunogenicity. here, we evaluated the basic immunogenic characteristics of an ad-hbv vaccine by monitoring the induced hbv-specific t cell responses in a glp three-month repeat dose toxicity study using cynomolgus monkeys as a non-human primate model. in general, as a new and extensively-accepted therapy option, the major toxicological effects observed possibly associated with the activation of the immune response induced by ad-hbv, for example erythema on the injection point skin and abnormalities of peripheral blood lymphocyte subsets. although there were transient increase in the other physiological parameters (wbc, crea, tp, alb, et al.) compared with vehicle control group, their values were still within normal ranges, and so they did not have toxicological significance. compared with vehicle control group, there were some significance (p< . or p< . ) in some parameters (cd + cd + t cell, cd +/cd +, cd + cd +) in low-dose, mid-dose and ad -null groups in different days, but it mostly was change in a single index and no dosing depended, so it was no biological significance. although the anti-adenovirus antibody were produced in almost all animals after first immunization and these the cut-off titer value was : . antibody titers of each group are respectively shown as purple (ad -null control), green (high-dose group), red (mid-dose group), and blue (low-dose group) curves. antibody titers were compared using a non-parametric test (friedman's test with dunn's multiple comparisons test); ** p value < . fig. neutralization of a cells with a luciferase-expressing adenovirus (ad -luc ). neutralization was determined by transgene expression inhibition in cells pretreated with mixed serum at different dilutions: : , : , and : . neutralization efficiency was calculated from triplicate measurements; quadrate measurements are shown for . × viral particles (vp) of ad -luc and × a cells per well. *p < . and **p < . antibody could partially neutralize the ability of adenovirus infection, ad-hbv still induced strong and broad ifnγ+ cell responses and displayed a whole array of immunogenic characteristics, which showed the highly immunogenic potential of ad-hbv vaccine. because adenovirus vectors are predominantly sequestered by the liver after administration, we analyzed the levels of ifn-γ and il- protein in the live by immunochemistry assay, and found that these two antiviral cytokines in the high-dose and ad -null control group were higher expressed than those in the vehicle control group. the current study has several limitations. first, natural killer (nk) cells naturally enriched in liver [ ] , and both of hbv infection [ ] and adenovirus vector delivery [ ] can affect the function of nk cells. however, we did not properly evaluate the function of nk cells in this study. in addition, it should be noted, we did not further investigate which cells targeted the hbv antigens/domains encoded by ad-hbv and produced the antiviral cytokines (ifn-γ and il- ). overall, this non-clinical safety assessment showed that the ad-hbv immunotherapeutic product induced no biologically significant toxicity, induced strong and broad ifnγ+ cell responses, and displayed a whole array of immunogenic characteristics, demonstrating the high immunogenic potential of the ad-hbv vaccine. moreover, this candidate vaccine has the advantage of gathering three hbv antigens in one vector together with the capacity to induce potent antiviral responses and broad immunogenic characteristics. thus, ad-hbv should be selected as a lead candidate for clinical development. high (%) ( %) ( %) ( %) *# ( %) *# ( %) *# il- low (%) ( %) ( %) ( %) ( %) ( %) high (%) ( %) ( %) ( %) *# ( %) *# ( %) *# * and # p < . compared with the vehicle control or low-dose group, respectively fig. representative images of positive immunohistochemical staining for il- , tnf-α, and ifn-γ in cynomolgus monkey liver tissues. images were taken 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recognition not applicable. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. additional file : table s . effects of ad-hbv administration on hematology. (pdf kb) additional file : table s . effects of ad-hbv administration on serum chemistry. (pdf kb) additional file : all data generated or analyzed during this study are included in this published article [and its supplementary information files.authors' contributions xz participated in the design of experiments, carried out the interpretation and analysis of in vitro and in vivo data, and helped to draft the manuscript. jw, jl and rl were involved in drafting the manuscript and participated in all experiments involving animals, including histological analyses. sz has been involved in all aspects of the study, including experimental design, analysis and interpretation of data, and manuscript writing. all authors read and approved the final manuscript. the animal experiments were conducted in a glp-compliant and aaalacaccredited laboratory (jiangsu tripod preclinical research laboratories co., ltd., nanjing, china), and all study protocols were approved by the testing facility's institutional animal care and use committee. the authors declare that there are no competing interest regarding the publication of this paper. key: cord- -igmb yfe authors: oma, veslemøy sunniva; tråvén, madeleine; alenius, stefan; myrmel, mette; stokstad, maria title: bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: igmb yfe background: bovine coronavirus (bcov) is a widely distributed pathogen, causing disease and economic losses in the cattle industry worldwide. prevention of virus spread is impeded by a lack of basic knowledge concerning viral shedding and transmission potential in individual animals. the aims of the study were to investigate the duration and quantity of bcov shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after bcov infection. methods: a live animal experiment was conducted, with direct contact between animal groups for h as challenge procedure. four naïve calves were commingled with a group of six naturally infected calves and sequentially euthanized. two naïve sentinel calves were commingled with the experimentally exposed group three weeks after exposure. nasal swabs, feces, blood and tissue samples were analyzed for viral rna by rt-qpcr, and virus isolation was performed on nasal swabs. serum was analyzed for bcov antibodies. results: the calves showed mild general signs, and the most prominent signs were from the respiratory system. the overall clinical score corresponded well with the shedding of viral rna the first three weeks after challenge. general depression and cough were the signs that correlated best with shedding of bcov rna, while peak respiratory rate and peak rectal temperature appeared more than a week later than the peak shedding. nasal shedding preceded fecal shedding, and the calves had detectable amounts of viral rna intermittently in feces through day and in nasal secretions through day , however virus isolation was unsuccessful from day six and day from the two calves investigated. viral rna was not detected in blood, but was found in lymphatic tissue through day after challenge. although the calves were shedding bcov rna days after infection the sentinel animals were not infected. conclusions: prolonged shedding of bcov rna can occur, but detection of viral rna does not necessarily indicate a transmission potential. the study provides valuable information with regard to producing scientifically based biosecurity advices. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. bovine coronavirus (bcov) is an important livestock pathogen with a high prevalence worldwide. the virus causes respiratory disease and diarrhea in calves and winter dysentery in adult cattle. these diseases result in substantial economic losses and reduced animal welfare [ ] . one way of reducing the negative consequences of this virus is to prevent virus transmission between herds. inter-herd transmission is possible either directly via transfer of live animals [ , ] , or indirectly via contaminated personnel or equipment [ ] . measures to prevent virus spread between herds must be based upon knowledge of viral shedding, the potential for transmission to susceptible animals and the role of protective immunity. several observational studies have been published on bcov shedding in feces of diarrheic calves and after transportation to feedlots [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, relatively few studies on bcov pathogenesis with emphasis on transmission potential under controlled conditions have been published. bcov belongs to the genus betacoronavirus within the family coronaviridae, also including the closely related hcov-oc , which causes respiratory infections in humans, and the human pathogens sars-cov and mers-cov [ ] [ ] [ ] . bcov consists of one serotype with some antigenic variation between different strains [ , ] . acutely infected animals develop antibodies that persist for a long period, possibly for several years [ ] [ ] [ ] . however, the protective immunity is shorter and incomplete. in two experimental studies, infected calves were not protected against reinfection with a different bcov strain three weeks after the first challenge, but did not develop clinical signs [ , ] . bcov is transmitted via the fecal-oral or respiratory route [ ] . it infects epithelial cells in the respiratory tract and the intestines; the nasal turbinates, trachea and lungs and the villi and crypts of the small and large intestine, respectively [ , ] . replication leads to shedding of virus in nasal secretions and in feces. important factors for the pathogenesis are still not fully explored, such as how the virus infects enterocytes shortly after introduction to an animal. viremia has been detected in one study by park et al. [ ] . clinical signs range from none to severe, and include fever, respiratory signs and diarrhea with or without blood [ , ] . as the time of infection is usually unknown and laboratory diagnostics are usually not performed, occurrence of clinical signs is the most relevant parameter to relate to viral shedding. the majority of experimental studies have used bcov inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. it has been hypothesized that bcov can cause chronic subclinical infections which could be an important virus source [ ] . kapil et al. documented viral antigen in the small and large intestines of infected calves three weeks post inoculation [ ] . crouch et al. found that ten cows were shedding bcov-immune complexes in the feces for weeks [ ] . it is, however, difficult to establish whether there is true persistence of virus, or reinfection of partially immune animals and whether these animals represent a risk to other animals. there is a lack of experimental studies investigating viral shedding pattern for longer periods than two weeks, with sensitive detection methods. viral load and infectivity also needs to be determined. this is of high practical relevance, since the farmers need guidance on biosecurity in trade and transport of live animals. the current study was conducted to fill prevailing gaps in the knowledge on fundamental aspects of bcov infection. the specific aims were to: a live animal experiment with the natural host was conducted. the experimental units were groups of calves and the intervention consisted of direct contact with bcov-infected animals. the primary outcome was clinical signs, and the secondary outcome was presence of bcov rna and bcov antibodies. three experimental groups were included; the field group (fg, n = ) that was naturally infected with bcov, the naïve exposed group (eg, n = ) and the naïve sentinel group (sg, n = ). an overview of the study design is shown in fig. . twelve bcov seronegative weaned bull calves between six and twelve weeks of age were included, seven were swedish red and white, four were swedish holstein and one swedish mountain breed. they originated from two dairy herds, initially negative for antibodies to bcov in milk from primiparous cows. the calves were allocated to groups according to herd of origin and day of arrival. the sequence of euthanasia of the eg and sg calves was random, determined by drawing of lots. natural outbreak of winter dysentery fg originated from a herd that was in an early phase of a winter dysentery outbreak. when fg was transported to the research facility, the calves showed mild signs of respiratory disease. two days later, a severe outbreak confirmed by rt-pcr and serology to be caused by bcov with bloody diarrhea and reduction in milk production, took place in the herd. the experiment was conducted at the stationary clinic at the department of clinical sciences at the swedish university of agricultural sciences. the facility was closed for other animals during the experiment, and had restricted admission for people. personnel used designated clothing, and had no contact with other cattle the same day. each group was housed in separate pens within the same room. due to the type of facility and design of the study, acclimatization period was not possible for any of the groups. clinical examinations and sampling were consistently done in the order sg, eg and fg. to mimic standard managerial conditions, direct contact was chosen as challenge procedure for both eg and sg. the commingling was done by moving eg into the other two groups' pens for h. efforts were made to minimize the stress and discomfort for the animals involved. the calves were kept groupwise in pens with straw bedding, were fed a commercial calf concentrate twice daily and had access to haylage ad libitum. the animals were monitored by a trained animal technician and a veterinarian at least three times a day. indications for antibiotic treatment ( iu procaine benzyl penicillin/kg bodyweight/day i.m. for five consecutive days) were abnormal sounds on lung auscultation or prolonged high temperature. indication for treatment with a non-steroidal anti-inflammatory drug (metacam vet, boehringer ingelheim vetmedica, germany) was severe depression, and oral fluid with electrolytes was to be given to moderately dehydrated animals. euthanasia was achieved by i.v. injection of pentobarbital (euthasol vet., le vet, netherlands). daily clinical examinations were performed by a veterinarian and clinical signs were scored as presented in fig. timeline of the experiment. the solid lines symbolize the timespan when the calves participated in the experiment. the dashed lines symbolize commingling of the indicated animals for h; e.g. the field group arrived at the research facility on day − , commingled with the exposed group day and left the research facility day . the calves in the exposed group were sequentially euthanized from day to day . the sentinel group arrived at the research facility day and commingled with the exposed group the following hours the score from each category was added to give a daily clinical score for each of the calves in the experiment score above six on three consecutive days as moderate disease and a score above eleven was categorized as severe clinical disease. nasal swab specimens and fecal samples from fg were collected approximately every third day from day − (d- ) to d . from eg, nasal swabs and fecal samples were collected every day from d to d and then every third day until d . nasal swabs from sg were collected d , d and d . the nasal specimens were collected by rotating a flocked eswab™ (copan, brescia, italy) approximately five cm inside one of the calf's nostrils. the specimens were frozen and stored at − °c before further processing. blood was drawn from the jugular vein upon arrival and d , d , d , d , d , d , d , d , d , d and d using sterile evacuated tubes with and without edta-anticoagulant. the edta-blood was centrifuged and the cell fractions were stored separately at − °c before further processing. sera were stored at − °c until analyzed. tissue samples from lung, medial retropharyngeal and mesenteric lymph nodes, ileum, and colon were stored in rna-later at − °c. serum samples were analyzed for anti bcov igg by svanovir bcv-ab (boehringer ingelheim svanova, uppsala, sweden) according to the manufacturer's instructions. samples from sg were also tested for antibodies to bovine respiratory syncytial virus (brsv) by svanovir brsv-ab (boehringer ingelheim). the optical density (od) at nm was measured and corrected by subtracting the od for the negative control. percent positivity (pp) was calculated as (sample od/positive control od) × , and a pp-value of < was regarded as negative. fecal samples (diluted : in pbs) and nasal swab specimens were centrifuged at x g for min. rna was extracted from μl supernatant and μl plasma by qiaamp viral rna mini qiacube kit (qiagen, hilden, germany), eluted in μl and frozen at − °c. rna from blood cell fractions from calf e on d and calf e on d was extracted with qiazol (qiagen) and chloroform phase separation mixed with % ethanol ( : ) and purified using rneasy mini kit column (qiagen), while rna was extracted from - mg tissue samples, using rneasy plus universal mini kit (qiagen). rt-qpcr was performed using rna ultrasense™ one-step quantitative rt-pcr system (invitrogen, ma, usa). two microliters of rna was added to a μl reaction volume containing nm each of forward and reverse primers and nm taqman probe [ ] . the thermal profile included an rt step with min at °c followed by °c for min. thereafter, cycles with s at °c and s at °c were conducted. the rt-qpcr was performed on a stratagene mx p™ (agilent technologies, ca, usa) and a positive and a negative control were included in each run. in order to evaluate inhibition of the rt-qpcr, rna extract from some fecal samples were diluted : and compared to undiluted rna. the ct-values in these samples suggested negligible levels of inhibitors. inhibitors in plasma and cell extracts were evaluated by spiking with mengovirus rna. comparison of ct-values showed that plasma had no negative effect, while the cell fractions had an inhibitory effect, giving an increase of one ct-value. in order to estimate the number of bcov viral rna copies (vrc) in the clinical samples, a standard curve was prepared using tenfold dilutions of a plasmid containing the bcov target sequence. aliquoted bcov rna was used as a calibrator and included in every rt-qpcr plate to adjust for inter plate variation. the number of vrc in the clinical samples was calculated using the formula: where q s = viral rna copies in sample, q c = viral rna copies of calibrator, ct s = ct value of sample, ct c = ct value of calibrator and m = slope of the standard curve. the standard curve covered the range from . to . × plasmid copies, and showed a strong linear relationship with a high coefficient of determination (r = . ) and a high amplification efficiency ( . %). the limit of quantification (loq) for the plasmid was . copies which represented . log bcov vrc per nasal swab and ml plasma, . log vrc/g feces and . log vrc/g tissue. virus infectivity was tested by virus isolation from nasal swabs from e and e between d and d (d , d , d , d , d , d , d , d and d ). the swab supernatants were diluted : in dulbecco's modified eagle medium (dmem, thermo fisher scientific, paisley, scotland), filtered through a . μm filter (sartorius stedim biotech, goettingen, germany) and added to a monolayer of -days-old human rectal tumor cells (hrt- g, attcc crl- ) in a -well plate. in addition, infective virus was titrated from one nasal swab supernatant using two-fold endpoint dilutions in a well plate. after h incubation at °c, the inoculum was replaced with dmem with % fetal calf serum and antibiotics ( iu penicillin and mg streptocillin/ ml). after two days at °c and % co , the cells were fixed with intracellular fixation buffer (ebiosience, ca, usa) and stained with : dilution of monoclonal mouse anti-coronavirus antibody labelled with fluorescein isothiocynate (biox diagnostics, rochefort, belgium) and dapi nuclear counterstain (thermo fischer scientific). the wells were observed under a fluorescent microscope for antigen positive cells. an overview of clinical signs in all groups is presented in table . five out of six fg calves showed mild clinical disease. eg's daily clinical scores are shown in fig. . three out of four eg calves showed mild disease, and one calf moderate clinical disease. sg did not develop clinical signs that were categorized as disease in the clinical scoring system. however, both calves had some days with intermittent nasal discharge and sporadic cough and s had a few days with intermittently runny feces. blood-tinged diarrhea or nasal discharge was not observed in any of the groups. all calves tested negative for antibodies to bcov at the beginning of the trial. at d all calves in fg and eg had seroconverted (additional file : table s ). the sg was still seronegative to bcov d and did not show an increase in titer for antibodies to brsv. bcov rna was not detected in any of the blood samples analyzed. the nasal shedding of bcov rna from fg and eg is presented in fig. a , and fig. shows eg calves' individual shedding. briefly, fg was shedding bcov rna d- through d , and in eg all swabs were positive from d through d , and at least one out of four calves was positive through d (fig. a) . two calves were positive in nasal swabs with a concentration of . log and . log vrc/swab the day of commingling with sg. none of the nasal swabs from sg were positive. fecal shedding of bcov rna in fg and eg is shown in fig. b , and the individual shedding from eg in fig. . viral rna was detected in fecal samples from fg between d- and d . fecal samples from eg were negative d and d . at least two out of four calves were positive every day from d through d and bcov rna was intermittently detected through d . after d , three calves had a period of four to six days with negative results, before they again started shedding bcov rna for three to five days (fig. ) . the association between bcov pcr results and selected clinical signs is shown in figs. and . the overall clinical score showed good correlation with detection of bcov rna. general depression and cough were the individual scores that showed the best association with bcov rna shedding. the highest mean respiratory rate and rectal temperature appeared more than a week later than the peak shedding. the calves were exposed to bcov in the field (f - ), were exposed to f-animals (e - ) or exposed to e-animals (s - ). a peak rectal temperature (rt) b runny to watery stools were considered diarrheic. c mucopurulent or purulent nasal discharge (nasal discharge score = ). d five days of i.m. treatment with iu procaine benzylpenicillin was initiated on indicated day. e calf f was treated for six days viral rna was detected in lymph nodes from the eg calves euthanized three, four, five and six weeks after infection (table ). viral rna was also detected in ileum and colon from the animals euthanized five and six weeks after infection, but not in lung tissue. virus was isolated from nasal swabs from calf e on d and from e in the period d to d . a photograph of infected cells is shown in fig. . the titer of infective bcov in the nasal swab was per μl swab medium ( ml in total) corresponding to . log infective particles in a swab containing . log vrc, giving a total to infective particles ratio (t/i) of log . the present study showed that calves infected with bcov shed viral rna for five weeks, and harbored viral rna in intestinal tissues and lymph nodes even longer. interestingly, contact with these calves three weeks after challenge, when the clinical condition had improved and the calves had seroconverted, did not lead to infection in sentinel calves and virus isolation was not possible from calves shedding viral rna at this time point. in concordance with other studies [ , ] , all eg calves became bcov positive shortly after contact with infected calves and shed viral rna continuously for two weeks. this supports that introduction of bcov into a naïve population leads to a high basic reproduction number (r ). r depends on the duration of the infectious period, the number of exposed susceptible individuals and the probability of a susceptible individual to be infected. in herds and transportation systems where cattle from different herds are commingled, the risk of virus transmission is high. the detection of bcov rna in nasal swabs from naïve calves in eg shortly after exposure might be due to passive inhalation of virus excreted by the fg, or to virus replication in the respiratory tract. since the viral load in the nasal swabs from eg exceeded that of fg at d , the study confirms that bcov replicated massively in the airways of eg calves already at d . fecal shedding started later than nasal shedding which is in concurrence with other studies [ ] . saif and colleagues found that when inoculating calves intranasally, bcov was first detected in nasal epithelial cells and secondly in feces. in contrast, in calves inoculated orally, fecal detection of bcov preceded detection in nasal swab specimens. they concluded that the infection route could determine the sequence of infection of the respiratory and intestinal tract [ ] . the present study supports that the respiratory route is the most common infection route when calves are naturally infected by direct contact. with indirect virus spread, the fecal-oral route could be more common. nasal swabs were more often positive for bcov than fecal samples in this trial, most likely due to a higher limit of detection for bcov in feces than in nasal swabs. for diagnostic purposes, nasal swab specimens therefore seem advantageous to fecal samples for virus detection in calves with suspected bcov related disease. moving and commingling are associated with stress, which has been found to affect the intestinal immune system [ ] . it is possible that stress increased the bcov rna shedding observed in the eg calves after introduction of the sentinel calves. buying and selling of calves often involve extended transportation and commingling with susceptible cattle. the stress response, and a possible increased fecal shedding of virus, would probably be higher under field conditions. shedding of virus in conventionally reared piglets, only in gnotobiotic piglets [ ] . this indicates that secondary pathogens and changes in microbiota are important for disease development and clinical signs. the present study supports that after the acute stage of disease other factors than virus replication are important for clinical signs; for instance secondary bacterial infections. although the sentinel calves did not get infected with bcov, they showed sporadic unspecific signs during the trial, but below the mildest category "mild disease" in the clinical scoring system. since acclimatization was not possible, the calves changed environment including feeding routines when enrolled in the experiment, which could cause the signs observed. other infectious agents could also have been present, and if so, most likely less virulent pathogens. bovine virus diarrhea virus and bovine herpesvirus are not present in sweden [ ] , and the sentinel calves showed no serologic response to brsv. co-infection between bcov and other agents is likewise possible in fg and eg, as is the case under field conditions. unlike most enteric viruses, bcov is enveloped and therefore susceptible to environmental inactivation [ ] . one might expect that the conditions in the forestomaches and abomasum would inactivate bcov and one possibility is that bcov is transported from the oronasal cavity to the small intestines through the bloodstream. however, viremia was not detected in the present study, and transport of the virus to the intestines appears to have been through the digestive tract. park and colleagues [ ] detected bcov rna in serum samples from calves infected with a winter dysentery strain between day three and eight post inoculation. they used nested pcr for detection, which is generally a more sensitive method than rt-qpcr, but also more vulnerable for contamination [ ] . short viremic period or intake of a lower virus dose in naturally infected calves could also explain the negative results in the present study. inhibition of the rt-qpcr by plasma components was tested and ruled out. despite the absence of detectable viremia in the present study, bcov rna was found in mesenteric lymph nodes at late stages of the infection. viral rna must have been transferred in low concentrations in blood or lymph to the draining lymph node, by antigen presenting cells or as free virus particles. the finding of bcov rna in lymph nodes, ileum and colon six weeks after infection indicates coronavirus persistence in calves, however, the importance of this persistence for virus transmission is uncertain. other coronaviruses are known to create persistent or chronic infections in mice and cats [ , ] . mers-cov is shown to be excreted for more than a month in humans [ ] and human coronavirus e creates persistent infections in vitro [ ] . although fecal shedding of bcov rna was detected five weeks post infection in the present study, the transmission potential at this stage is most likely negligible, as at three weeks post infection. bcov vrc were quantified by rt-qpcr, which does not give information on the number of infective particles. the ratio of total to infective particles (t/i) is challenging to establish for bcov due to difficulties in cultivating virus from clinical samples. in the present study, virus titration showed a t/i ratio of approximately tissue samples from lymph nodes, lung, ileum and colon were harvested from exposed group calves euthanized at the indicated number of days after exposure to field group calves. the number of viral rna copies (vrc) of bcov was quantified with rt-qpcr and the limit of quantification was . log vrc/g tissue log . with this high t/i ratio it is not surprising that virus isolation was unsuccessful after d , when the vrc numbers are decreasing. it also agrees with the sentinel calves not getting infected d . in contrast, roughly . log vrc were detected per nasal swab and gram feces from the seronegative fg calves that infected the eg calves. with a t/i ratio of log , each nasal swab and gram of feces contained more than . log infective virus particles. the high t/i ratio and the failure of virus isolation after d could be due to either few infective particles or low sensitivity of the isolation method. low levels of infective particles could be caused either by high production of defective particles or by neutralizing effect of antibodies. low sensitivity could be caused by suboptimal conditions in cell culture compared to in vivo (particularly for virus from clinical samples not adapted to cell culture growth), dilution of viral content in the swab, and freezing and thawing of the material. for feline enteric coronavirus, the t/i increased from - log during the first week after infection, to up to log days post infection [ ] , the increase possibly caused by the antibody response. few methods are available for studying transmission potential apart from live animal experiments, although ethically challenging and resource demanding. existing literature is based on experimental studies examining bcov shedding for [ , , ] to [ , ] days. to the authors' knowledge, the present study is the first to study the shedding for as long as six weeks under experimental conditions. in addition, it is also the first to study the impact of this shedding using sentinel calves. although a low number of calves were used, the results indicate that calves are not infectious three weeks after exposure to bcov. this information is important and relevant in order to produce scientific based advices on how to avoid introduction of bcov into herds. further investigation of calves at different stages of disease is recommended to verify and corroborate these findings. the effect of stress related to transport on viral shedding and infectivity should also be considered. in the present study, the virus that caused winter dysentery in adult cattle primarily gave respiratory disease in calves. niskanen et al. also found that bcov derived from an outbreak of winter dysentery caused mainly respiratory disease in weaned calves [ ] , supporting that bcov is an important cause of respiratory disease in calves [ , ] and winter dysentery in adults [ ] . the economic and welfare consequences of bcov therefore include the combined effects of neonatal enteritis, respiratory disease in young cattle and winter dysentery in adults. also considering the high prevalence worldwide, bcov is an important loss-inflicting factor in the cattle industry. the current study shows that calves infected with bcov are rt-qpcr positive in nasal and fecal specimens for a longer period than earlier recognized. however, contact with naïve calves three weeks after exposure did not lead to infection. a low level of infective particles could be due to either production of a high level of defective particles and/or production of neutralizing antibodies. the study provides highly relevant information when designing biosecurity advice regarding animal trade and coronaviral disease in cattle. additional file : table s . antibodies to bcov. 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vso, mt, sa, mm and ms conceived and designed the experiment; vso, sa and mt performed the experiment; vso and mm planned and performed the lab analyses. all authors wrote, read and approved the manuscript. the authors declare that they have no competing interests. the trial was conducted in line with national and international guidelines for the care and use of animals and approval was given by the ethics committee for animal experiments, uppsala, sweden [protocol number c / ].• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: